Host Cells and Methods for Producing Diacid Compounds

ABSTRACT

The present invention provides for a method of producing one or more fatty acid derived dicarboxylic acids in a genetically modified host cell which does not naturally produce the one or more derived fatty acid derived dicarboxylic acids. The invention provides for the biosynthesis of dicarboxylic acid ranging in length from C3 to C26. The host cell can be further modified to increase fatty acid production or export of the desired fatty acid derived compound, and/or decrease fatty acid storage or metabolism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority as a continuationapplication to PCT International Patent Application No.PCT/US2011/061900, filed Nov. 22, 2011, which claims priority to U.S.Provisional Patent Application Ser. No. 61/416,287, filed Nov. 22, 2010,both of which are herein incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The invention described and claimed herein was made utilizing fundssupplied by the U.S. Department of Energy under Contract No.DE-AC02-05CH11231. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention is in the field of production of dicarboxylicacids or diacid compounds derived from fatty acids, and in particular,host cells that are genetically modified to produce fatty acid-deriveddiacids.

BACKGROUND OF THE INVENTION

Aliphatic dioic acids, alcohols and compounds having combinations ofalcohols and acids are versatile chemical intermediates useful as rawmaterials for the preparation of adhesives, fragrances, polyamides,polyesters, and antimicrobials. While chemical routes for the synthesisof long-chain α,ω)-dicarboxylic acids are available, the synthesis iscomplicated and results in mixtures containing dicarboxylic acids ofshorter chain lengths. As a result, extensive purification steps arenecessary. Chemical synthesis is the preferred route of synthesis forthese compounds today.

Picataggio reports conversion of the dodecane (a C12 linear alkane) andtetradecane (a C14 linear alkane) or their corresponding fatty acids(dodecanoate, tetradecanoate) into their corresponding α,ω-diacarboxylicacids using the yeast Candida tropicalis (see, e.g., Picataggio, et al.,Biotechnology 10:894-898, 1992). The method described is greatlydisadvantaged by its reliance upon exogenous addition of C12 or C14alkane, or C12 or C14 fatty acid; moreover, the method is disadvantagedby the inability of Candida to convert other, non C12 or C14, fatty acidand alkane substrates into corresponding diacids. Thus, a method for theendogenous production of fatty acid substrates of desired chain lengthand subsequent omega oxidation of the substrates, producing thecorresponding ω-hydroxy fatty acid or α,ω-dicarboxylic acid, wouldprovide an economical, competitive route to valuable α,ω-dicarboxylicacids, ω-hydroxy fatty acids, diamines, etc that has no precedence.

Thus, there remains a need for methods and materials for biocatalyticconversion of feedstock chemicals into their corresponding ω-hydroxyfatty acids and α,ω-diacarboxylic acids, methods for producing theω-hydroxy fatty acid and α,ω-diacarboxylic acid in a fermentation broth,methods for controlling the ω-hydroxy fatty acid or α,ω-diacarboxylicacid or fatty acid chain length, methods for secreting or retaining theproduct from/in the cells, and methods for purifying the product fromthe culture broth. The present invention meets these needs.

SUMMARY OF THE INVENTION

This present invention provides recombinant host cells and relatedmethods and materials for the biocatalytic production ofα,ω-dicarboxylic acids, ω-hydroxy fatty acids, fatty acids (FA), orother fatty acid-derived molecules from fermentable carbon sources andprovides a source of diacids for the production of renewable chemicalsfor use in applications, including making polyesters, resins,polyamides, nylon, fuel additives and fuels, lubricants, paints,varnishes, engineering plastics and the like.

The invention provides host cells and methods for producing fatty acids,ω-hydroxy fatty acids, α,ω-dicarboxylic acids, and related compoundswith controlled chain lengths from inexpensive feedstocks, includingcornstarch, cane sugar, glycerol, and other carbon sources. Theinvention also provides methods for making specific short and long chainfatty acids, diacids, and diols that have not previously been made bybiosynthetic methods in microbial host cells.

In nature there exist multiple routes for microbial production of fattyacids of different chain lengths. The most abundant in nature are thefatty acid pathways, of which there are three primary systems: the TypeI, Type II, and Type III fatty acid systems. Type I and Type III fattyacid systems often contain multiple enzymatic activities on a singlepolypeptide chain and are referred to as elongases for the Type IIIsystem. Generally, Type I and Type III systems generate specific chainlength acyl-CoA molecules, which are normally transferred directly intothe production of membrane lipids (phospholipids, glyerolipids, etc.)but can be hydrolyzed by a thioesterase to release the free fatty acidin engineered systems. Type II fatty acid systems are composed of singlepolypeptides that individually encode the multiple enzymatic activitiesrequired for fatty acid biosynthesis to generate a range of fattyacyl-ACPs that are normally transferred directly into the production ofmembrane lipids, but can be hydrolyzed by a thioesterase that recognizesspecific chain length fatty acids.

Certain cells also make molecules called polyketides that containaliphatic backbones similar to fatty acids. Certain of these polyketidesare made by Type I polyketide synthases (PKSs). Type I PKSs are composedof catalytic modules that minimally contain an acyl carrier protein(ACP), acyl transerfase (AT), and a ketosynthase (KS) and in someinstances contain a ketoreductase (KR), a KR and a dehydratase (DH), anda KR, DH, and an enoyl reductase (ER). Type I PKSs generally contain athioesterase (TE) to cleave the product from the acyl-ACP thioester,unlike natural fatty acid systems that directly incorporate acyl-ACPs oracyl-CoAs through transfer reactions. In Type I PKSs, the startermolecule and the total number of extension modules dictates the lengthof the final product. Type I PKSs' modular nature has made them amenableto engineering a variety of products not made by naturally occurringPKSs.

The enzymatic decarboxylation of a 2-keto acid substrate results in theformation of the corresponding aliphatic aldehyde; subsequent oxidationof the aldehyde to the corresponding carboxylic acid produces thecorresponding fatty acid. All cells make a variety of 2-keto acids asintermediates in amino acid biosynthesis. Cells engineered foroverexpression of native or engineered enzymes encoded by genes in theLeuABCD operon (for example, and without limitation, in E. coli) extendby a single carbon the 2-keto acid substrate 2-ketobutyrate into longerchain length 2-keto acids.

In accordance with the methods of this invention, engineered cells andrecombinant vectors are provided in which Type I, II, III fatty acidsynthases, Type I PKSs, and 2-ketoacid biosynthesis pathways,decarboxylases, and oxidases are genetically engineered to make freefatty acids of a specific chain length.

In various embodiments of the host cells and recombinant DNA vectors ofthe present invention, Type I PKS systems are engineered to produce aspecific chain length fatty acid by choosing the appropriate number ofmodules that terminate with a thioesterase that cleaves the thioesterbond and releases a free carboxylate. This thioesterase can becovalently attached to the PKS polypeptide, or expressed independently.In other embodiments of the present invention, a recombinant Type Ifatty acid system is employed to produce fatty acid. The FA biosynthesisenzymes produce a specific chain length acyl-thioester, and athioesterase is used to produce a fatty acid for subsequent oxidation tothe dicarboxylic acid. In other embodiments of the invention, a type IIfatty acid system is employed to produce fatty acid, thioesterasesspecific for desired chain lengths are employed to produce the desiredchain length fatty acid product. In other embodiments of the invention,a Type III fatty acid system is employed to produce specific chainlength fatty acid coenzyme A (CoA) esters, and a thioesterase isemployed to cleave the specific chain length fatty acid from CoA. Inother embodiments of the present invention, a Type I hybrid PKS systemis employed to produce a desired chain length fatty acid, where theC-terminal PKS domain is a thioesterase that cleaves the fatty acid fromthe acyl carrier protein. In yet other embodiments of the presentinvention, a 2-keto acid pathway, 2-keto acid decarboxylase, andaldehyde dehydrogenase are used to produce a desired chain length fattyacid.

Numerous microbes can be employed for the production of fattyacid-derived chemicals in accordance with the methods of the invention.In various embodiments, the microbes have characteristics that allowthem to produce higher levels of product. For example, in oneembodiment, the host organism provided by the invention lacks or hasreduced expression levels of, or has been modified for decreasedactivity of, enzymes catalyzing the degradation of specific chain lengthfatty acids. These enzyme activities include CoA-ligases (for example,and without limitation, FadD (E. coli), FAA1, FAA2, FAA3, FAA4 (S.cerevisiae), etc as provided later and enzymes necessary for betaoxidation of fatty acids (for example, and without limitation, POX1,POX2, IDP3, TES1, FOX3 (S. cerevisiae), etc as provided later). In someembodiments of the present invention, diols are produced from fattyacids. In these embodiments, enzymes necessary for beta oxidation willbe reduced, but CoA-ligases may be retained.

Because malonyl-CoA is an essential precursor to fatty acid synthesis,it is advantageous to upregulate malonyl-CoA biosynthesis. In variousembodiments of the invention, the host organism has been engineered forincreased expression of enzymes catalyzing production of malonyl-CoA.For example, and without limitation, increasing the expression level ofactyl-CoA carboxylase (gene ACC1 (FAS3) in S. cerevisiae is includedherein for reference).

Thus, the invention provides a variety of different engineered hostorganisms that exhibit improved production of fatty acids and thecorresponding diacid products. In various embodiments of the invention,the host organisms have reduced expression of genes and/or theircorresponding enzyme products associated with fatty acid,α,ω-dicarboxylic acid, and related product, beta-oxidation, and haveincreased expression of genes and/or their corresponding enzyme productsassociated with α,ω-dicarboxylic acid and related product transporters.In this manner, the organism is deficient in its ability to degrade thefinal fatty acid or α,ω-dicarboxylic acid product and/or secretesproduct into the fermentation broth. Furthermore, the organism has beenengineered for increased expression of genes and/or their correspondingenzyme products associated with biosynthesis of malonyl-CoA. In someembodiments, the methods of the invention are practiced with host cellsin which the genes/enzymes that promote storage of fatty acids and soimpede the ability to achieve high production levels of a given fattyacid derived product have been inactivated or engineered to reduceexpression level/activity.

In some embodiments, the host organism is yeast. Yeast host cellssuitable for practice of the methods of the invention include, but arenot limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces,Schizosaccharomyces and Pichia, including engineered strains provided bythe invention. In one embodiment, the yeast host cell is a species ofCandida, including but not limited to C. tropicalis, C. maltosa, C.apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii,C. intermedia, C. lipolytica, C. panapsilosis and C. zeylenoides. In oneembodiment, Candida tropicalis is the host organism.

In some embodiments the host is bacteria. Bacterial host cells suitablefor practice of the methods of the invention include, but are notlimited to, Escherichia and Bacillus, including engineered strainsprovided by the invention. In one embodiment, the bacterial host cell isa species of Bacillus, including but not limited to B. subtilis, B.brevis, B. megaterium, B. aminovorans, and B. fusiformis. In oneembodiment, B. subtilis is the host organism.

In the methods of the present invention, once a fatty acid of a desiredchain length is produced, it is hydroxylated at the omega carbon toproduce a ω-hydroxy fatty acid. In many embodiments, the hydroxylationis achieved by expressing a cytochrome P450 or monooxygenase that isspecific for hydroxylation at the terminal (omega, ω-) carbon of a fattyacid (EC 1.14.15.3). ω-hydroxy fatty acids are themselves valuable andare used in the production of fast drying paints and varnishes, etc. Assuch, the methods of the invention provide that, if desired, theω-hydroxy fatty acid can be isolated. Alternatively, the omega-hydroxyfatty acid is then, in accordance with the methods of the invention,further oxidized to a α,ω-dicarboxylic acid. In various embodiments, theroute of oxidation is through an aldehyde, mediated by either a fattyalcohol dehydrogenase (FAD) or fatty alcohol oxidase (FAO) (these twoterms are used interchangeably; EC 1.1.3.20 or 1.1.3.13). The aldehydeintermediate is then converted into a diacid by an aldehydedehydrogenase (ADH; EC 1.2.1.3 or 1.2.1.4). In some embodiments, theP450 monoxygenase carrying out hydroxylation of the omega carbon is P450BM3 from B. megaterium, either wild type or engineered for alteredregiospecficity; for example, without limitation, introduction ofmutation of phenylalanine 87 to alanine (mutation F87A) in P450 BM3alters enzyme regiospecificity toward increased hydroxylation of fattyacid substrates at the omega position (Oliver et al, Biochemistry,36:1567-1572, 1997).

FIG. 1 shows various biosynthetic reactions provided by the method ofthe invention. Using the Type I, II, or III fatty acid synthase by themethod of the invention, a desired fatty acid is producedintracellulary, hydroxylated by a cytochrome P450 at the omega carbon,and then enzymatically oxidized to the α,ω-dicarboxylic acid. Using theType I PKS by the method of the invention, a desired fatty acid isproduced from the PKS system by appropriate selection of the acyl-CoAloading module, extension modules, and thioesterase; the resulting fattyacid is subsequently hydroxylated by a fatty acid omega hydroxylase (EC1.14.15.3), and oxidized to the corresponding α,ω-dicarboxylic acid byan alcohol oxidase (EC 1.1.3.20 or 1.1.3.13) and aldehyde dehydrogenase(EC 1.2.1.3 or 1.2.1.4).

FIG. 4 shows a 2-keto acid-based pathway to production of theα,ω-dicarboxylic acid adipic acid. By the method of the invention,2-ketoheptanoate is produced from the naturally occurring substrate2-ketobutyrate in the host organism through the activity of enzymesencoded by the LeuABCD operon genes. 2-ketoheptanoate is subsequentlydecarboxylated to 1-hexanal throught the activity of the KivDdecarboxylase, oxidized to the fatty acid by aldehyde dehydrogenase (EC1.2.1.3), to the ω-hydroxy fatty acid by a fatty acid omega hydroxylase(EC 1.14.15.3), and to the α,ω-dicarboxylic acid adipate by an alcoholoxidase (EC 1.1.3.20 or 1.1.3.13) and aldehyde dehydrogenase (EC 1.2.1.3or 1.2.1.4).

Thus, the invention provides new pathways for making α,ω-dicarboxylicacids in modified host cells. While all yeast, E. coli, and Bacillushosts have endogenous routes to production of the α,ω-dicarboxylic acidsuccinate, no other α,ω-dicarboxylic acids are produced in unmodifiedhost cells. In one aspect, the present invention provides a method forproducing one or more fatty acid-derived dicarboxylic acid compounds ina genetically modified host cell that does not naturally produce theα,ω-dicarboxylic acid compounds. For example, and without limitation,yeast and E. coli hosts do not make α,ω-dicarboxylic acids by themethods of this invention. Bacillus is not known to naturally produceany α,ω-dicarboxylic acids, except pimelic acid, by the methods of theinvention; furthermore, the methods of the invention provide additionalroutes to other diacids in a Bacillus host.

In one aspect, the present invention provides methods for thebiosynthesis of fatty acid derived α,ω-dicarboxylic acids compoundsranging in carbon length from C3 to C26, including both even and oddnumbers of carbons. Such α,ω-dicarboxylic acid compounds include, butare not limited to, C3 diacids, C4 diacids, C5 diacids, C6 diacids, C7diacids, C8 diacids, C9 diacids, C10 diacids, C11 diacids, C12 diacids,C13 diacids, C14 diacids, C15 diacids, C16 diacids, C17 diacids, C18diacids, C19 diacids, C20 diacids, C21 diacids, C22 diacids, C23diacids, C24 diacids, C25 diacids, and C26 diacids.

In other embodiments of the invention, the methods for producingω-hydroxy fatty acids are provided. In these embodiments of theinvention, appropriate selection of the P450 enables hydroxylation ofthe free fatty acid at the ω-position. For example, and withoutlimitation, expression of native P450 BM3 results in mixed hydroxylationof numerous fatty acid substrates at the ω-1, ω-2 and ω-3 positions;introduction of the point mutation F87A into the P450 BM3 amino acidsequence imparts ω-hydroxylation regioselectivity when using variousfatty acid substrates. As described in the preceding paragraph, suchω-hydroxy fatty acid compounds include, but are not limited to, C3 toC26 ω-hydroxy fatty acids.

One can modify the expression of a gene by a variety of methods inaccordance with the methods of the invention. Those skilled in the artwould recognize that increasing gene copy number, ribosome binding sitestrength, promoter strength, and various transcriptional regulators canbe employed to alter an enzyme expression level. The present inventionprovides a method of producing a fatty acid derived α,ω-dicarboxylicacid compounds in a genetically modified host cell that is modified bythe increased expression of one or more genes involved in the productionof fatty acid compounds, such that the production of fatty acidcompounds by the host cell is increased. The invention also providessuch genetically modified host cells. Such genes include, withoutlimitation, those that encode the following enzymatic activities: acetylCoA carboxylase, ketosynthase, ketoreductase, dehydratase, enoylreductase, cytosolic thiosterase, and acyl-carrier protein. Illustrativegenes that encode these enzymatic functions include acpP, acpS, accA,accB, accC, accD, fabD, fabH, fabG, fabZ, fabA, fabI, fabB, fabF(suitable copies of these genes may be obtained from, and withoutlimitation, E. coli, B. subtilis), tesA, tesB (E. coli), yneP, ysmA,ykhA, yvaM, ylpC (B. subtilis), FAS1, FAS2, FAS3, ELO1, ELO2, ELO3 (S.cerevisiae), ELO1, ELO2, ELO3 (T. brucei, T. cruzi, L. major), fasA,fasB (C. glutamicum, B. ammoniagenes, C. ammoniagenes), FAS1 (Mycoplasmatuberculosis, Mycoplasma. smegmatis), and hexA, hexB (A. flavus, A.parasiticus). In other embodiments, one increases transcriptionalregulation of these genes. Suitable transcriptional regulators includefadR (suitable copies of these genes may be obtained from, and withoutlimitation, E. coli or B. subtilis) and RAP1, ABF1, REB1, INO2, INO4 (S.cerevisiae).

The present invention also provides a method of producing a fatty acidderived α,ω-dicarboxylic acid compound in a genetically modified hostcell that is modified by the decreased or lack of expression of one ormore genes encoding proteins involved in the storage and/or metabolismof fatty acid compounds, such that the storage and/or metabolism offatty acid compounds by the host cell is decreased. Such genes include,without limitation, the following: the acyl-CoA sterol transferases ARE1(S. cerevisiae), ARE2 (S. cerevisiae), diacylglycerol acyl transferases,DGA1 (S. cerevisiae) and LRO1 (S. cerevisiae), plsB, plsX (E. coli),yhfL, lcfA, des, plsX, cypC, and yhfT (B. subtilis) genes.

The present invention also provides methods and host cells that havebeen engineered to be capable of secreting or excreting the product intothe media. In one embodiment, engineered host cells and methods areprovided to make fatty acids that are secreted or excreted into thefermentation broth. In particular embodiments, these geneticallymodified host cells are modified by expression of one or more genesencoding proteins involved in the export of α,ω-dicarboxylic acid, fattyacid, or ω-hydroxy fatty acid compounds such that the product is movedfrom the interior of the cell to the exterior. Such genes include thefollowing: DAL5, DIP5, JEN1 (S. cerevisiae), MAE1 (Schizosaccharomycespombe), atoE, citT (B. subtilis), dcuB, dcuC (B. subtilis, A.succinogenes, E. coli), and various multidrug resistance pumps.

Once in the fermentation broth, the diacids and hydroxy acids can beseparated and purified in accordance with the invention. In variousembodiments of the invention, the microbe is engineered to secrete fattyacids, α,ω-dicarboxylic acids, or ω-hydroxy fatty acids and subsequentlypurified from the broth. In various embodiments of the invention, theproducts are purified through precipitation as calcium salts, orreactive extraction with tertiary amines. In various embodiments of theinvention, the tertiary amines employed include, and without limitation,tripropylamine, trioctylamine, or tridecylamine. In some embodiments ofthe invention, ion exchange is employed for further purification of thefatty acid, α,ω-dicarboxylic acid, or ω-hydroxy fatty acid.

In other embodiments, the host cells are not engineered or modified tosecrete the product into the growth medium and the product accumulatesin the host cell. In these embodiments, the diacid product is separatedfrom the host cell in accordance with the invention by centrifugation orsettling of the cell material, cell lysis, and subsequent purificationof the diacid product as described above.

Thus, the present invention further provides for a wide variety ofgenetically modified host cells useful in practice of the methods of thepresent invention. In various embodiments, the host cell is geneticallymodified in any one of and any combination of the genetic modificationsdescribed herein.

The present invention further provides for an isolated dicarboxylic acidcompound produced from the methods of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and others will be readily appreciated by theskilled artisan from the following description of illustrativeembodiments when read in conjunction with the accompanying drawings.

FIG. 1 illustrates five of the general α,ω-dicarboxylic acid productionmethods of the present invention. A summary of the engineered pathwaysfor production of fatty acids from Type I, Type II, and Type III FAS,Type I PKS, and 2-ketoacid synthesis is provided. Fatty acids of thedesired chain length are produced using Type I, Type II, or Type IIIfatty acid synthase systems or Type I hybrid PKS systems, ordecarboxylation and oxidation of 2-ketoacids. The resulting fatty acidis subsequently oxidized to the corresponding α,ω-dicarboxylic acid byfatty acid a fatty acid omega hydroxylase (EC 1.14.15.3), fatty alcoholoxidase (1.1.3.20 or 1.1.3.13), and aldehyde dehydrogenase (1.2.1.3 or1.2.1.4). FIG. 1 depicts a progression that shows the flow of carbonfrom a feedstock such as sugar, through a FA node to form specific chainlength FAs, which are then oxidized at the ω carbon to produceω-hydroxy-FAs, ω-oxo-FAs, and finally, α,ω-dicarboxylic acids. Enzymesare italicized and major producted are indicated in bold.

FIGS. 2A and 2B illustrate FT-MS analysis of strains producing diacidsfrom co-cultures expressing LtesA and one of two P450s. Strain BM3 is anengineered E. coli host DH1 ΔfadD expressing P450-Bm3 and P450-Bm3(F87A); strain LtesA-Bm3 is an engineered E. coli host DH1 ΔfadDexpressing LtesA, P450-Bm3, and P450-Bm3 (F87A). Strains were analyzedfor production of fatty acid, ω-hydroxyacid, and the α,ω-dicarboxylicacid. (See, Example 1 below). FIG. 2A is compiled data from MS analysisto identify tetradecanoic acid, 13- or 14-hydroxy-tetradecanoic acid,and tetradecanedioic acid from cultures expressing P450 Bm3 alone orcultures coexpressing a thioesterase, LtesA and P450 Bm3s. There is nodetectable product for cultures expressing Bm3 alone, but there isproduction of tetradecanoic acid, 13- or 14-hydroxy-tetradecanoic acid,and tetradecanedioic acid in cultures expressing the P450 Bm3 and LtesA.FIG. 2B is data showing the identification of the molecular ion fortetradecanedioic acid from the MS data for cultures expressing both theP450 Bm3 and LtesA.

FIG. 3 illustrates the plasmids that were used in Example 1, below. E.coli DH1 ΔfadD was employed; cultures were grown for 24 h in TB mediawith 1 mM IPTG at 30° C., sampled, and analyzed for production of fattyacid, ω-hydroxyacid, and the α,ω-dicarboxylic acid.

FIG. 4 illustrates use of a 2-ketoacid pathway for the production of anα,ω-dicarboxylic acid (e.g., adipate) in accordance with an embodimentof the invention. Similar short-chain α,ω-dicarboxylic acids can beproduced by varying the 2-ketoacid overproduced in the host cell.Following decarboxylation of the fatty acid with substrate promiscuousKivD decarboxylase, or other related decarboxylases (EC 4.1.1.X), theresulting fatty acid is subsequently oxidized to the correspondingα,ω-dicarboxylic acid by a fatty acid omega hydroxylase (EC 1.14.15.3),alcohol oxidase (1.1.3.20 or 1.1.3.13), and aldehyde dehydrogenase(1.2.1.3 or 1.2.1.4).

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail, it is to be understoodthat, unless otherwise indicated, this invention is not limited toparticular nucleic acids, expression vectors, enzymes, hostmicroorganisms, or processes, as such may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “expressionvector” includes a single expression vector as well as a plurality ofexpression vectors, either the same (e.g., the same operon) ordifferent; reference to “cell” includes a single cell as well as aplurality of cells; and the like.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings.

The terms “optional” or “optionally” as used herein mean that thesubsequently described feature or structure may or may not be present,or that the subsequently described event or circumstance may or may notoccur, and that the description includes instances where a particularfeature or structure is present and instances where the feature orstructure is absent, or instances where the event or circumstance occursand instances where it does not.

The terms “host cell” and “host microorganism” are used interchangeablyherein to refer to a living biological cell that can be (or has been)transformed via insertion of an expression vector. Thus, a host organismor cell as described herein may be a prokaryotic organism (e.g., anorganism of the kingdom Eubacteria) or a eukaryotic cell. As will beappreciated by one of ordinary skill in the art, a prokaryotic celllacks a membrane-bound nucleus, while a eukaryotic cell has amembrane-bound nucleus.

As used herein, a “recombinant cell” or “recombinant host cell” refersto a host cell that has been genetically altered to comprise aheterologous nucleic acid sequence. Such a heterologous sequence may be:(i) an exogenous nucleic acid that is not native to the cell, e.g., anexogenous gene, an exogenous promoter, an optimized coding sequence, amutated coding sequence; (ii) extra copies of an endogenous gene orpromoter; (iii) or nucleic acids, e.g., a promoter operably linked to acoding region, that are heterologous to one another. It is understoodthat such terms refer not only to the particular subject cell but to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “heterologous nucleic acid” or “heterologous DNA” as usedherein refers to a polymer of nucleic acids wherein at least one of thefollowing is true: (a) the sequence of nucleic acids is exogenous to(i.e., not naturally found in) a given host microorganism (b) thesequence may be naturally found in a given host microorganism, but in anunnatural (e.g., greater than expected) amount; or (c) the sequence ofnucleic acids comprises two or more subsequences that are not found inthe same relationship to each other in nature. For example, regardinginstance (c), a heterologous nucleic acid sequence that is recombinantlyproduced will have two or more sequences from unrelated genes arrangedto make a new functional nucleic acid. For example and withoutlimitation, the present invention describes the introduction of anexpression vector into a host microorganism, wherein the expressionvector contains a nucleic acid sequence coding, e.g., a promoter and/orcoding region, that is not normally found in a host microorganism. Withreference to the host microorganism's genome, then, the nucleic acidsequence is heterologous.

The terms “expression vector” or “vector” refer to a nucleic acidcompound and/or composition that transduces, transforms, or infects ahost microorganism, thereby causing the cell to express nucleic acidsand/or proteins other than those native to the cell, or in a manner notnative to the cell. An “expression vector” contains a sequence ofnucleic acids (ordinarily RNA or DNA) to be expressed by the hostmicroorganism. Optionally, the expression vector also comprisesmaterials to aid in achieving entry of the nucleic acid into the hostmicroorganism, such as a virus, liposome, protein coating, or the like.The expression vectors contemplated for use in the present inventioninclude those into which a nucleic acid sequence can be inserted, alongwith any preferred or required operational elements. Further, theexpression vector must be one that can be transferred into a hostmicroorganism and replicated therein. Preferred expression vectors areplasmids, particularly those with restriction sites that have been welldocumented and that contain the operational elements preferred orrequired for transcription of the nucleic acid sequence. Such plasmids,as well as other expression vectors, are well known to those of ordinaryskill in the art.

The term “transduce” as used herein refers to the transfer of a sequenceof nucleic acids into a host microorganism or cell. Only when thesequence of nucleic acids becomes stably replicated by the cell does thehost microorganism or cell become “transformed.” As will be appreciatedby those of ordinary skill in the art, “transformation” may take placeeither by incorporation of the sequence of nucleic acids into thecellular genome, i.e., chromosomal integration, or by extrachromosomalintegration. In contrast, an expression vector, e.g., a virus, is“infective” when it transduces a host microorganism, replicates, and(without the benefit of any complementary virus or vector) spreadsprogeny expression vectors, e.g., viruses, of the same type as theoriginal transducing expression vector to other microorganisms, whereinthe progeny expression vectors possess the same ability to reproduce.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free of components that normally accompanyit in its native state.

As used herein, the terms “nucleic acid sequence,” “sequence of nucleicacids,” and variations thereof shall be generic topolydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base,and to other polymers containing normucleotidic backbones, provided thatthe polymers contain nucleobases in a configuration that allows for basepairing and base stacking, as found in DNA and RNA. As used herein, thesymbols for nucleotides and polynucleotides are those recommended by theIUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022,1970).

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter) and asecond nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

In some embodiments, the invention provides for a method for producing aα,ω-dicarboxylic acid in a genetically modified host cell, the methodcomprising: culturing a genetically modified host cell under a suitablecondition to produce enzymes in a system to oxidize fatty acids toω-hydroxy fatty acids to α,ω-dicarboxylic acids. In some embodiments,such a genetically modified host cell comprises first a enzyme thatproduces a fatty acyl-CoA (or acyl-ACP), a second optional enzyme thatis a thioesterase, an fatty acid omega oxidase (EC 1.14.15.3) thatoxides the fatty acid at the ω-carbon to produce a ω-hydroxy fatty acid,a second oxidase, i.e., a fatty alcohol dehydrogenase (FAD) or fattyalcohol oxidase (FAO) (these two terms are used interchangeably; EC1.1.3.20 or 1.1.3.13), to oxidize the ω-hydroxy fatty acid to analdehyde, and an aldehyde dehydrogenase (ADH; 1.2.1.3 or 1.2.1.4) toconvert the aldehyde into an α,ω-dicarboxylic acid. Again, the methodsof the invention are generally illustrated in FIG. 1 and involve eitherType I, II, or III fatty acid synthase systems, or a Type I PKS systemor a 2-ketoacid system, wherein the carbon length of the output fattyacid is controlled. FIG. 4 illustrates the extension of endogenouslyproduced 2-ketybutyrate substrate to longer chain 2-ketoacids throughtthe activity of enzymes encoded by the LeuABCD operon, subsequentdecarboxylation to the fatty aldehyde, and oxidation to the fatty acid.In various embodiments, appropriately selected oxidizing enzymes performomega oxidation on said fatty acid.

In some embodiments, the genetically modified host cell comprises afirst nucleic acid construct encoding the first enzyme (i.e., theelongase), optionally a second nucleic acid construct encoding thethioesterase, a third nucleic acid encoding the fatty acid omegahydroxylase, a fourth nucleic acid encoding the FAD or FAO and a fifthnucleic acid encoding the ADH, and the culturing results in theexpression of the elongase, (optionally) the thioesterase, the fattyacid omega hydroxylase, the FAD or FAO and the ADH.

In some embodiments, the method further comprises the step of recoveringthe diacid produced, wherein the recovering step is concurrent orsubsequent to the culturing step. Kurzrock et al, report on multiplepurification strategies used for isolation of the microbially produceddiacid succinate from fermentation broth (Kurzrock et al. BiotechnologyLetters, 32:331-339, 2010); these methods, including precipitation withcalcium hydroxide, calcium oxide, or ammonia; electrodialysis; reactiveextraction with long chain aliphatic primary, secondary, or tertiaryamines (for example, and without limitation, tripropylamine,trioctylamine, or tridecylamine) in organic solvent; and ion exchangeare generally applicable for purification of fatty acid,α,ω-dicarboxylic acid, ω-hydroxy fatty acids products. In variousembodiments of the invention, the products are purified throughprecipitation as calcium salts, or reactive extraction with tertiaryamines. In various embodiments of the invention, the tertiary aminesemployed include, and without limitation, tripropylamine, trioctylamine,or tridecylamine. In some embodiments of the invention, ion exchange isemployed for further purification of the fatty acid, α,ω-dicarboxylicacid, or ω-hydroxy fatty acid.

In various embodiments, the method comprises a method of geneticallymodifying a cell, e.g., a bacterial or yeast cell, to increaseexpression of one or more genes involved in the production of fatty acidcompounds; such that the production of fatty acid compounds by the cellis increased. Such genes encode proteins such as acetyl-CoA carboxylase(ACC), cytosolic thiosterase (LtesA), a fatty acid synthase, andacyl-carrier protein (AcpP). In some embodiments, the geneticallymodified cell may be modified to produce higher levels of cytosolicacetyl-coA and malonyl-CoA. Thus, in some embodiments a geneticallymodified cell may comprise a modification to express, or increaseexpression of, proteins such as ATP citrate lyase.

In various embodiments, the genetically modified host cell expresses anenzyme system for producing α,ω-dicarboxylic acids from a simple sugarsubstrate (for example, but not limited to, glucose, sucrose, xylose,arabinose; such sugars might be obtained from cornstarch, sugar cane,cellulosics, and waste biomass), wherein the enzyme system comprises: anelongase to produce a fatty-acyl-CoA-thioester of a desired chainlength; a fatty acid omega hydroxylase (EC 1.14.15.3) to hydroxylate thefatty acid at the omega carbon to produce a ω-hydroxy fatty acid; anoxidase to oxidize the ω-hydroxy fatty acid to an aldehyde (EC 1.1.3.20or 1.1.3.13); and an aldehyde dehydrogenase to produce theα,ω-dicarboxylic acid (EC 1.2.1.3 or 1.2.1.4); and wherein at least oneof the enzymes are recombinant enzymes encoded by one or more expressioncassettes.

In various embodiments, the genetically modified host cell expresses anenzyme system for producing α,ω-dicarboxylic acids from a simple sugarsubstrate (for example, but not limited to, glucose, sucrose, xylose,arabinose; such sugars might be obtained from cornstarch, sugar cane,cellulosics, and waste biomass) wherein the enzyme system comprises: anelongase to produce a fatty-acyl-CoA-thioester of a desired chainlength; a thioesterase that produces a fatty acid from theacyl-thioester; an oxidase to hydroxylate the fatty acid at the omegacarbon to produce a ω-hydroxy fatty acid (EC 1.14.15.3); an oxidase tooxidize the ω-hydroxy fatty acid to an ω-oxo fatty acid (EC 1.1.3.20 or1.1.3.13); and an aldehyde dehydrogenase to produce the α,ω-dicarboxylicacid (EC 1.2.1.3 or EC 1.2.1.4), wherein at least two of the enzymes arerecombinant enzymes encoded by one or more expression cassettes.

Enzymes and Constructs Encoding Thereof

As noted above, one of the advantages of the present invention is thatit does not rely upon exogenous alkanes, fatty acids (FA), or hydroxy-FAsupplementation or the ability of the microbe to produce enoughsubstrate for conversion into diacids (or other products). Instead, inthe methods of the present invention, the fatty acid starting materialis also microbially produced. Numerous methods for microbially producingfatty acids are known to those of skill in the art and include thosemethods described in PCT International Publication Nos. WO 2007/136762,WO 2008/100251 and WO 2010/075483 as well as those methods described inU.S. Patent Application Publication No. US 2010/0170148, the teachingsof all of which are incorporated herein by reference. Both Type I, II,and III FASs and Type I PKS are employed in various embodiments of themethods of the present invention.

While the invention provides modified host cells, methods and enzymesfor the production of fatty acid molecules via five distinct routes butwithout limitation and including Type I, II and III fatty acid, Type IPKS and 2-ketoacid biosynthetic pathways, first we focus on enzymesinvolved in fatty acid biosynthesis. Table 1, below, provides Type I,II, and III fatty acid synthases and elongases and other enzymesinvolved in the biosynsthesis of fatty acids suitable for use oralteration in accordance with the methods and in the host cells of theinvention.

In Table 1 below we provide suitable enzymes, without limitation, forperforming the methods in accordance with the invention that are used toproduce fatty acids via Type I, II or III fatty acid biosynthesis. Indetail, the “enzyme” column provides both the gene, enzyme name and itsaccession number either in NCBI, Genbank, UniProt, or associatedcatalytic activity, unless the gene name is unavailable in which caseonly enzyme function and accession numbers are provided. The“modification” column describes the genetic modification in accordancewith the invention; “OE” means overexpress and in some embodiments ofthe invention, where the host cell does not have an endogenous copy ofthe gene it is taken to mean the enzyme is expressed heterologously. Inother embodiments, in which the host cell has an endogenous copy of thegene the gene product is overexpressed. Express and overexpress meanthat enzyme levels and activity are increased compared to the wild-typecase and those skilled in the art appreciate that this can be achievedby increasing the strength or changing the type of the promoter,increasing the strength of the ribosome binding site or Kozak sequence,increasing the stability of the mRNA transcript, altering the codonusage, and increasing the stability of the enzyme, etc. In themodification column, “decrease” means that the enzyme activity isdecreased compared to the wildtype. Those skilled in the art willappreciate that decreasing enzyme activity compared to wildtype isachieved in a variety of ways in accordance with the methods of theinvention and not limited to completely removing an enzyme by geneknockout, addition of an inhibitor compound that reduces or eliminatesenzyme's activity, expression level is modulated such that total enzymeactivity is decreased by weakening a promoter, ribosome binding site orKozak sequence, by decreasing mRNA transcript stability or by increasingprotein degradation. The “use” column indicates a more specific use ofthe enzyme without limitation in accordance with the methods of theinvention and in some cases indicates the fatty acid chain lengthproduct. For example, the “hexA” enzyme is involved in producing a fattyacid chain six carbons in length and this is indicated by “C6”. The“organism” column indicates suitable sources for the genes and enzymesand does not necessarily indicate the choice of host cells. Finally,superscripted numbers indicate relevant citations.

TABLE I ENZYMES INVOLVED IN FATTY ACID SYNTHESIS OE = overexpress;Organism = an illustrative, non-limiting organism that is a source ofthe gene/enzyme Mod- Gene names that encode ifica- enzyme tion UseOrganism FAS3 (Acetyl-CoA OE C3 S. cerevisiae carboxylase; NP_014413.1)FAS2 (α-subunit of fatty OE C16, C18 S. cerevisiae acid synthase;NP_015093.1) FAS1 (β-subunit of fatty OE C16, C18 S. cerevisiae acidsynthase; NP_012739.1) ACB1 (acyl-coA binding OE Sequesters S.cerevisiae protein; NP_011551.1) fatty acyl- CoAs ELO1 (elongase I; OEC10 T. brucei ¹³ XP_824876) ELO1 (XP_813972 OE C10 T. cruzi Beta ketoacyl synthases & OE C10-C22 L. Major elongases (CAJ02963, CAJ02967,CAJ02975, CAJ02982, CAJ02986, CAJ03003, CAJ03006, CAJ03013, CAJ03016,CAJ03023, CAJ03028, CAJ03035, CAJ02037, CAJ08636) ELO2 (elongase II; OEC22 S. cerevisiae NP_009963) ELO2 (elongase II; OE C14 T. brucei ¹³XP_824877) ELO3 (elongase III; OE C26 S cervisiae NP_013476) ELO3(elongase III; OE C18 T. brucei ¹³ XP_824878) ELO4 (elongase IV; OE C22T. brucei ¹³ XP_824041) HexA (fatty acid synthase OE C6 A. flavus, II;AF391094) A nomius HexA (fatty acid synthase OE C6 A. parasiticus, II;AF391094) A. flavus HexB (fatty acid synthase I; OE C6 A. nomiusAY510454) FAS2 (fatty acid synthase OE C6 A. parasiticus II; AY371490)ERG 10 (thiolase; NP_015297) OE C4 S. cerevisiae atoB (thiolase;NP_416728) OE C4 E. coli phaA (thiolase; YP_353824) OE C4 R.sphaeroides, R. eutropha phaB (acetoacetyl-CoA OE N/A R. solanacearumreductase; NC_014307) Hbd (acetoacetyl-CoA OE N/A C. beijerinckii,reductase; YP_001307783) A. caviae Crt (crotonase; NP_891288) OE N/A C.beijerinckii phaJ (crotonase;) OE N/A P. stutzeri, YP_004715374) T.denticola Bcd/etfA-B OE N/A C. acetobutylicum (3hydroxybutyryl-CoAdehydrogenase; NP_349154, NP_349155, NP_349314 fabH (ketosynthase; OEN/A E. coli NP_415609) fabHB (ketosynthase; OE N/A B. subtilisNP_388898) fabHA (ketosynthase; OE N/A B. subtilis NP_389015) fabA(3hydroxydecanoyl- OE N/A B subtilis, ACP dehydrase; E. coli NP_415474,NP_388285.2) fabZ (3hydroxydecanoyl- OE N/A B subtilis, ACP dehydrase;E. coli NP_391518.2) fabI (enoyl reductase; OE N/A B subtilis,NP_389054.2, E. coli NP_415804.1) fabB (ketosynthase; OE N/A B subtilis,NP_416826.1, E. coli NP_389016.1) fabF (ketosynthase; OE N/A B subtilis,NP_389016.1, E. coli NP_415613.1) fabG (ketoreductase; OE N/A Bsubtilis, NP_389473.1, E. coli NP_389732.1, NP_389732.1, NP_390820.1,NP_415611.1, fabD (malonyl-CoA OE N/A B subtilis, transacylase;NP_389472.1, E. coli NP_415610.1) acpP (acyl carrier protein; OE N/A Bsubtilis, NP_389474.1, E. coli NP_415612.1) acpS (phosphopantetheinyl OEN/A B subtilis, transferase; NP_417058.1, E. coli NP_388343.1) DVU2560(YP_011772.1) OE N/A D. vulgaris fabH (ketosynthase; OE N/A D. vulgarisYP_011773.1) acpP (acyl carrier protein; OE N/A D. vulgaris YP_011774.1)fabF (ketosynthase; OE N/A D. vulgaris YP_011775.1)

Non-engineered cells typically produce a range of fatty acid chainlengths with varying degrees of saturation to maintain membranefluidity, etc and usually rely on acyl-transferases to move the fattyacid from the FAS into products that compose cell membranes likediacylglycerols and phospholipids. Under typical conditions, they do notutilize thioesterases as part of the fatty acid biosynthetic machineryand in the case where a fatty acid thioesterase may be present in anaturally occurring organism's genome, they often contain signalpeptides that target their expression to areas where fatty acidbiosynthesis is not occurring. Therefore, to use the thioesterases inaccordance with the methods of the invention, one skilled in the artwill appreciate the requirement to express the thioesterase in the samelocation as where the fatty acids are produced or located. In detail,TesA, a thioesterase native to E. coli has a leader peptide sequencethat targets its expression to the periplasm and to use thisthioesterase in accordance with the methods of the invention, thesequence must be removed to target its activity to the cytosol(indicated by LTesA) in the case of E. coli, as that is the sight offatty acid biosynthesis. However, in general, and in the methods inaccordance with this invention, thioesterases can be used for cleavingfatty acid moieties whenever the fatty acid is covalently attached via athioester bond to an acyl-carrier protein and this occurs in most TypeII FAS proteins as well as in Type I PKS proteins, while Type I and IIIFAS proteins typically generate a CoA bound fatty acyl thioester. Thedistinction here is emphasized because in some cases, the CoA thioesteris labile and the fatty acid can be released without a thioesterase,yet, in the other cases, a TE is required to efficiently cleave thethioester bond and release the fatty acid. Although natural hydrolysismay occur in the case of Type I and III FAS proteins (whose compositiondetermines chain length), the rates of hydrolysis in some in bodimentsis increased by expressing thioesterases. Another distinction is madebetween PKS TEs and FAS TEs, because often PKS TEs are incorporated intothe PKS polypeptide at the C terminal domain, whereas often FAS TEs areseparate proteins, although in the case of FASs this is not a rule.Because PKS TEs are typically this final domain, they will be discussedin a different section, but in some embodiments, a suitable TE is theDEBS TE from the erythromycin PKS pathway. TEs have selectivity forcleaving fatty acyl-CoA or fatty acyl-ACP thioester bonds exist innature and have been found in a variety of hosts, including but notlimited to plants, bacteria and eukaryotes. In accordance with themethods of the invention, for Type I, II, and III fatty acidbiosynthesis, a TE with appropriate fatty acid carbon chain lengthselectivity is chosen for a particular free fatty acid product. WhileType I, II, and III FASs and Type I PKSs enzymes utilize TEs, Table IIbelow provides illustrative thioesterases suitably used for the fattyacid synthase systems herein and in accordance with the methods of theinvention. To reiterate, in general, many thioesterases are availablefor use in connection with the fatty acid syntheses, a thioesterase maybe used, for example, to produce a fatty acid from either an ACP boundfatty acyl-thioester or to produce a fatty acid from a CoA bound fattyacyl-thioester. Typically with Type II FAS, a thioesterase is employedto cleave the ACP-bound fatty acid. With Type I & III FAS, thefatty-acyl-CoA-thioester is naturally hydrolyzed by water, therebyproviding the fatty acid and does not necessarily require athioesterase.

Thioesterases suitable for use in accordance with the methods of thepresent invention include those set forth in Table II below. The“thioesterase” column includes the enzyme name and accession number (inmost cases) in various forms; the “modification” column is defined aspreviously, including the operative definition of “OE” or overexpress;substrate specificity refers to the fatty acid chain length recognizedby the thioesterase; the “organism” column contains an illustrativeorganism that is suitable for obtaining the genetic element/enzyme, butis not mean to be limiting.

TABLE II Thioesterases Substate Thioesterases Modification specificityOrganism TesA (NP_415027.1) OE C10-C18 E. coli TesB (NP_414986.1) OEC6-C18 E. coli TES OE C12-C18 R. sphaeroides TES OE C6-C18 R.sphaeroides EST2 OE C6 A. acidocaldarius ESTA OE C6 A. acidocaldariusUcFATB1 (Q41635) OE C12 U. californica chFATB2 (AAC49269) OE C8, C10 C.hookeriana chFATB3 (AAC72881.1) OE C14 C. hookeriana

In addition to use of thioesterases in the fatty acid pathways describedabove, the PKS pathways also use thioesterases that in most cases are apart of the PKS peptide located at the C-terminus in accordance with themethods of the invention and are provided in the following. Analternative way of producing the fatty acid of a specific chain lengthin accordance with the invention is to employ a hybrid PKS. Exemplarymodules are listed herein. To make a fully reduced fatty acid of a givenchain length, one constructs, in accordance with the invention, a hybridthat contains a loading module (KS, AT, ACP) an extension module (KS,KR, DH, ER, ACP), and a thioesterase (TE). The choice of loading moduleand choice of extension modules that condense precursors likemalonyl-CoA or methylmalonyl-CoA determines whether the fatty acid chainis even or odd carbon number and the number of extension modulespreceding the TE determines the overall chain length. To construct anodd-chain fatty acid PKS in accordance with the invention, one selects aloading module that incorporates propionate via methylmalonyl-CoA, andto construct an even-chain fatty acid PKS, one selects a loading modulethat incorporates acetate via malonyl-CoA. Another method of theinvention involves selection of modules that incorporate longer chainacyl-CoA molecules like butyryl-CoA. Illustrative loading, extension,and thioesterase modules suitable for use in the methods, PKS, and hostcells of the invention are provided in the following, where a PKS knownto produce a specific compound is named followed by parantheticals thatidentify the source organism for the genetic material.

Non-limiting examples of loading modules for malonyl-CoA are provided asfollows: Niddamycin PKS (S. caelestis), Amphotericin PKS (Streptomycesnodosus), Concanamycin a PKS (Streptomyces neyagawaensis), EpothilonePKS (Sorangium cellulosum), Mycolactone PKS (Mycobacterium ulcerans),Nanchangmycin PKS (Streptomyces nanchangensis), Nystatin PKS(Streptomyces noursei), Oleandomycin PKS (Streptomyces antibioticus),Oligomycin (Streptomyces avermitilis), Pimaricin PKS (Streptomycesnatalensis), Pyoluteorin PKS (Pseudomonas fluorescens), stigmatellin PKS(Stigmatella aurantiaca).

Non-limiting examples of loading modules for methylmalonlyl-CoA areprovided as follows and are used in accordance with the methods of theinvention to load an odd-carbon number onto the PKS, but does notnecessarily require the final fatty acid product to be an odd-carbonnumber as described previously: Megalomicin PKS (Micromonosporamegalomiceas), Methymycin PKS (Streptomyces venezuelae), Monensin PKS(Streptomyces cinnamonensis), Narbomycin PKS (Streptomyces venezuelae),Neomethymycin PKS (Streptomyces venezuelae), Pikromycin (Streptomycesvenezuelae), Spinosad PKS (Saccharopolyspora spinosa), Tylactone PKS(Streptomyces fradiae).

An illustrative example of a loading module for propionyl-CoA is fromthe erythromycin PKS (Saccharopolyspora erythraea).

Non-limiting extension modules that incorporate malonyl-CoA viacondensation, increase the chain length by two carbons, and fully reducethe acyl chain are provided as follows, where “M” and the numberindicate the module number within the PKS post loading module, such that“M1” would directly follow a loading module in a given PKS sequence:Nystatin PKS M5, M15 (S. caelestis); Amphotericin PKS M5, M16(Streptomyces nodosus); Mycolactone PKS M9 (Mycobacterium ulcerans);Nanchangamycin PKS M6, M8 (Streptomyces nanchangensis); Oleandomycin PKSM3 (Streptomyces antibioticus); Stigmatellin PKS M5 (Stigmatellaaurantiaca); Soraphen PKS M2, M3, M5 (Sorangium cellulosum); MonensinPKS M6, M8 (Streptomyces cinnamonensis); Spinosad PKS M2(Saccharopolyspora spinosa); Herbimycin A PKS M6 (S. hygroscopicus);FROO8 PKS M19 (Streptomyces sp. FR-008)

An illustrative example of a thioesterase from the erythromycin PKS(Saccharopolyspora erythraea), which is sometimes referred to as theDEBS TE, is suitable for cleaving the fatty acyl ACP thioester bondproduced via PKSs and result in production of a specific chain lengthfatty acid.

Four major routes to producing a desired chain length fatty acid viaTypes I, II, or III FASs or Type I PKSs have been described in detail. Afifth route, described below, is through 2-ketoacid intermediates as ina portion of the leucine biosynthetic pathway.

Thus, in another aspect, the present invention provides methods forproducing fatty acids, ω-hydroxyacids, and α,ω-dicarboxylic acids (e.g.,adipic acid), using elements of amino acid biosynthetic pathways(2-ketoacid system). Normally cells do not produce fatty acids,ω-hydroxyacids, or α,ω-dicarboxylic acids from amino acid pathways. Innon-engineered cells, pyruvate in the tricarboxylic acid cycle isconverted into oxaloacetate, which is then converted through multiplesteps into L-threonine, and then into 2-ketobutyrate via a threoninedeaminase. 2-ketobutyrate is normally a substrate in cells for producingisoleucine and leucine, but has also been demonstrated to be a suitablesubstrate for elongation in one-carbon increments with an engineeredLeuA enzyme and native LeuBCD enzymes to produce the 2-ketovalerate(C5), 2-ketocaproate (C6), and 2-ketoheptanoate (C7) intermediates.Suitable mutations of the LeuA enzyme include, but are not limited to,G462D, S139G, H97A, N167A. These ketoacid intermediates are thendecarboxylated by a promiscuous enzyme, Kivd (from Lactococcus lactis)to form an aldehyde (see, Zhang, et al., “Expanding metabolism forbiosynthesis of non-natural alcohols, Proc Natl Acad Sci USA 105,20653-20658 (2008), the teachings of which are incorporated herein byreference). Suitable mutations of the KIVD enzyme include, but are notlimited to V461A, F381L.

In accordance with the teaching of the methods of this invention, weconvert the fatty aldehyde into a fatty acid by oxidation or expressionof an aldehyde dehydrogenase (EC. 1.2.1.3). In other embodiments of theinvention, we produce a fatty alcohol by expressing an alcoholdehydrogenase (ADH) that converts the aldehyde into an alcohol andserves as a substrate for oxidation to the fatty acid by methodsdescribed elsewhere. In some embodiments, a suitable, but not limited toalcohol dehydrogenase is ADH6 (S. cerevisiae). The pathway utilized inthis embodiment of the invention is illustrated in FIG. 4. Examples ofshort chain aldehyde dehydrogenases (ALDs) have been described andsuitable enzymes are listed below. Further oxidation of the omega-carbonis necessary, once the short chain fatty acid is produced, and isachieved through omega oxidation described in a following section.

To produce a fatty acid from the 2-ketoacid pathway, the supply of theketo acid (e.g., 2-ketobutyrate) is important as a precursor to thepathway, thus in some embodiments the invention provides cells that areengineered to produce appropriate substrate levels by overexpressinggenes that encode enzymes in the pathway. The present invention providesa number of ways to supply or increase the supply of 2-ketobutyrate,which ultimately increases the fatty acid, ω-hydroxyfatty acid, orα,ω-dicarboxylic acid product; these include, but are not limited to:threonine degradation pathways, isoleucine biosynthesis pathways (viacitramalate synthase and 2-methylmalate), glutamate pathways (via2-methylaspartate and 2-methyloxaloacetate) or χ-elimination ofo-phosphohomoserine and o-acetyl-homoserine. Other host cells andmethods of the invention exploit prevention of transamination ofketoacids by deletion or attenuation of various genes including, e.g.,ilvE, tyrB, etc.

Here, we provide enzymes without limitation in accordance with themethods of the invention for producing fatty aldehydes, fatty acids,w-hydroxy fatty acids, and diacids from 2-ketoacid precursors. First, weprovide the enzymes involved in elongating the 2-ketoacid precursor,2-ketobutanoate to 2-ketovalerate, 2-ketocaproate, and 2-ketoheptanoate,and decarboxylating these precursors to fatty aldehydes as providedbelow in Table III, with column definitions as previously described, butwith the “EC number” that provides the biochemical reaction associatedwith the provided enzymes.

TABLE III 2-ketoacid enzymes Enzyme EC Number Modification Organism leuA2.3.3.13 Overexpress: E. coli, B. subtilis G462D, S139G, H97A, N167AleuB 1.1.1.85 OE E. coli, B. subtilis leuC 4.2.1.33 OE E. coli, B.subtilis leuD 4.2.1.33 OE E. coli, B. subtilis tyrB 2.6.1.42 attenuateE. coli, B. subtilis ilvE 2.6.1.42 attenuate E. coli, B. subtilis KivdOE Lactococcus lactis ilvA 4.2.1.16 OE E. coli tdcB 4.2.1.16 OE E. coliCHA1 OE S. cerevisiae ILV1 4.3.1.9 OE S. cerevisiae LEU1 4.2.1.33 OE S.cerevisiae LEU2 1.1.1.85 OE S. cerevisiae BAT1, BAT2 attenuate S.cerevisiae

Having provided the enzymes for production of fatty aldehydes from2-ketoacid pathways in accordance with the methods of the invention, wenow provide without limitation enzymes for the conversion of the fattyaldehydes into fatty acids by expressing aldehyde dehydrogenase (ALD)enzymes performing biochemistry described by EC 1.2.1.3 and shown inFIG. 4. In general, many ALDs exist, but here we provide suitable,non-limiting examples by enzyme name and in paranthesis, an illustrativesource organism for the enzyme, and any associated specificity as acarbon chain length range (eg C4-C14): Ald1 (Acinetobacter sp M1;C4-C14); ScAld1 (Mus musculus; C6-C9); Psdr1 (Homo sapiens; C2-C12);ALD4 (S. cerevisiae; C2-C12), etc. In accordance with the methods of theinvention, we have now provided five routes to produce fatty acids withspecific carbon chain lengths via the Type I, II, and III FASs, Type IPKSs, and the 2-ketoacid biosynthetic enzymes. In the following, weprovide enzymes for the production of ω-hydroxyacids andα,ω-dicarboxylic acids from these fatty acid precursors, but next wedescribe production of other valuable chemicals from the 2-ketoacidpathways.

In addition to the α,ω-dicarboxylic acids, ω-hydroxyacids, diols andshorter chain monoacids are synthesized using the 2-keto acid pathway inaccordance with other embodiments of the invention. For instance, in oneembodiment, production of a monoacid is achieved by eliminatingreactions EC 1.14.15.3 and EC 1.1.3.20 from the pathway illustrated inFIG. 4. In another embodiment, production of a ω-hydroxyacid is achievedby eliminating reaction EC 1.1.3.20 from the pathway illustrated in FIG.4. In yet another embodiment, production of a diol is achieved byreplacing the ALD (aldehyde dehyrogenase) EC 1.2.1.3 with an aldehydereductase EC 1.1.1.21 in the pathway illustrated in FIG. 4.

The methods of the present invention involve the use of an oxidase tohydroxylate the fatty acid at the omega carbon to produce a ω-hydroxyfatty acid. As discussed in the background section above, for exampleCandida tropicalis shows the oxidation of the C12 and C14 fatty acid tothe C12 and C14 ω-hydroxy fatty acid is described, for example, byPicataggio, et al. (Biotechnology 10:894-898, 1992) and in U.S. Pat.Nos. 7,405,063; 7,160,708; 7,109,009; 7,063,972; 7,049,112; 6,790,640;and 6,331,420 as well as in PCT International Publication No. WO2004/013336, the teachings of all of which are incorporated herein byreference. In one embodiment, productivity of the {tilde over(ω)}oxidation is enhanced by amplification of both the cytochrome P450monooxygenase and NADPH- or NADH-cytochrome reductase genes or by usinghighly active promoters with such genes.

Once the fatty acid of a desired chain length is produced with one ofthe five routes it is hydroxylated in accordance with the invention atthe omega carbon, producing a ω-hydroxyfatty acid. ω-hydroxyfatty acidsthemselves are valuable and used in the production of rapidly dryingpaints and varnishes, etc.

Here, we provide without limitation and in accordance with the methodsof the invention enzymes that are suitable for overexpressing in theprovided host cells and results in hydroxylating the omega carbon toproduce ω-hydroxy fatty acids, i.e., an omega hydroxy fatty acid. The ECnumber describing the biochemical reaction that converts a fatty acidinto a ω-hydroxy fatty acid is EC 1.14.15.3. We provide non-limitingexamples of suitable enzymes by their name and in parentheses provide anillustrative organism from which to source the genetic material,followed by fatty acid chain length specificity where C3-C10 indicatesactivity on fatty acids ranging in chain length from three to tencarbons, e.g. “Enzyme Name” (“Source Organism”; Chain lengthspecificity). Any superscripts indicate references that describe theenzyme. Suitable enzymes for performing omega hydroxylation are asfollows: P450a1k1 (C. Tropicalis; C12-C16)^(2,9,10); CPR (C.tropicalis)²; P450 (3P2) (chimeric enzyme; C6-C12)¹¹; P450 (pHP3)(Rabbit; C6-C12)¹¹; P450 (P. oleovarans; C8-C12); P450 BM3 (B.megaterium; C12-C18); CYP86A8 (A. thaliana; C12-C18); CYP703A1 (Petuniax hybrida; C12) CYP704B2 (O. sativa ssp japonica; C18); CYP4V2 (H.sapiens; C12-C16); CYP4B (H. sapiens; C7-C10)¹²; CYP4A (H. sapiens;C10-C16)¹²; and CYP4F (H. sapiens; C16-C26)¹². Although here and in allembodiments for α,ω-dicarboxylic acid production we employ an omegahydroxylase, in some embodiments hydroxylating other carbons within thefatty acid or diacid backbone may be useful and can be accomplished byhydroxylases in general. In one embodiment, a P450 BM3 that has an F87Amutation is used to change the regiospecificity of hydroxylation anddemonstrates hydroxylation at the ω-1, ω-2, and ω-3 positions.

It will be readily apparent to those of skill in the art in view of thisdisclosure that ω-hydroxyfatty acids, i.e., 1-hydroxyfatty acids, arethemselves valuable and used in the production of rapidly drying paintsand varnishes, etc. As such, if of interest, the ω-hydroxyfatty acids,i.e., 1-hydroxyfatty acids, can be isolated or recovered in accordancewith the invention.

The ω-hydroxyfatty acids, i.e., ω-hydroxyfatty acids, can be, in otherembodiments, further oxidized to an α,ω-diacarboxylic acid. This omegahydroxy fatty acid can be further oxidized in accordance with theinvention to a diacid using methods described herein. We provide fattyalcohol oxidases (FAOs) or fatty aldehyde dehydrogenases (FADs) toconvert an omega-hydroxy fatty acid into an omega-oxo fatty acid inaccordance with the biochemical reaction EC 1.1.3.20. In one embodiment,the fatty alcohol oxidase provided is FAO1, FAO2a or FAO2b from Candidatropicalis. In one embodiment, the route of oxidation is through analdehyde using a FAO.

The aldehyde intermediate is then converted, in accordance with theinvention, into a diacid by an aldehyde dehydrogenase (ALD). In general,many ALDs exist. The following are non-limiting examples of suitableenzymes by enzyme name and in parentheses, an illustrative sourceorganism for the enzyme, and any associated specificity as a carbonchain length range (e.g., C4-C14): Ald1 (Acinetobacter sp M1; C4-C14);ScAld1 (Mus musculus; C6-C9); Psdr1 (Homo sapiens; C2-C12); ALD1, ALD2,ALD3, ALD4, ALD5, ALD6, HFD1 (S. cerevisiae; C2-C12).

Most cells naturally have the capacity to degrade fatty acids, hydroxylfatty acids and diacids to some capacity through enzymatic activitiesassociated with the β-oxidation pathway. Briefly, the pathway functionsin most cases by activating free fatty acid groups to CoA thioesterswith acyl-CoA ligases, which are further oxidized and degraded,proceeding through a 2,3 enoyl-CoA, 3-hydroxyacyl-CoA, 3-ketoacyl-CoA,and then to a two carbon-shortened acyl-CoA that repeats the cycle. Theenzymatic activity required for this degradation is known. In accordancewith the methods of this invention, we provide cells that have reducedor eliminated degradation pathways for fatty acids, hydroxyl fattyacids, and diacids compared to their wildtype counterparts. In someembodiments, the host organism is engineered in accordance with theinvention to remove or attenuate genes encoding fatty acyl-CoAsynthetase enzymes. In other embodiments, the host organism isengineered to remove or attenuate genes encoding acyl-CoAdehydrogenases. Methods for making host cells that are substantiallyβ-oxidation pathway blocked are known to those of skill in the art.Here, and in accordance with the methods of the invention, we providewithout limitation illustrative enzymes involved in fatty aciddegradation that are removed or attenuated to increase fatty acid,hydroxyl fatty acid, or diacid production in an engineered host. Indetail, we provide the enzyme name and in parantheticals its function.Superscripts provide references for certain enzymes. We provide hostcells with the following enzymes removed or attenuated in S. cerevisiaeor related yeasts that increase fatty acid, diacid or hydroxyl fattyacid production: ANT1 (adenine nucleotide transporter); POX2 (3hydroxyacyl-CoA dehydrogenase); IDP3 (isocitrate dehydrogenase); POX1(acyl-CoA oxidase); FOX3 (oxoacyl thiolase); EHD3 (hydrolase); PAST andPAS2 (peroxisomal formation protein); FAA1, FAA2, FAA3, and FAA4(acyl-CoA synthetase). We provide host cells with the following enzymesremoved or attenuated in E. coli: FadD and FadK (acyl-CoA synthetase);FadE and YdiO (acyl-CoA dehydrogenase); FadB, FadJ, and PaaZ (enoyl-CoAhydratase/hydroxyacyl dehydrogenase); FadA (3-ketoacyl thiolase); FadI(acetyl-CoA acyltransferase). We provide host cells with the followingenzymes removed or attenuated in B. subtilis or related yeasts thatincrease fatty acid, diacarboxylic acid or hydroxy fatty acidproduction: YhfT, YhfL, LcfA, YdaB, YtcL, and BioW (acyl-coAsynthetase); YdbM, YngJ, mmgC, acdA, and FadE (acyl-CoA dehydrogenase);YngF, YsiB, YhaR, and fadN (enoyl-CoA hydratase).

In addition, the host cell is, in some embodiments of the invention,genetically modified so that it has decreased or lacks expression of oneor more genes encoding proteins involved in the storage and/ormetabolism of fatty acid compounds, such that the storage and/ormetabolism of fatty acid compounds by the host cell is decreased. Suchgenes include the following: the ARE1, ARE2, DGA1, and LRO1 genes. Otherengineered host cells with genes that are modified are provided inaccordance with the methods of the invention and include those set forthin Table IV. The “enzyme” column provides the name of the enzyme to bemodified; the “manipulation” column provides the modification to theenzyme that is provided and is either “attenuate” or “OE”. Hereattenuate means either decreasing the enzyme activity or completelyeliminating it; “OE”=overexpress. The superscripts refer to references.

TABLE IV Genes Involved in Storage/Metabolism of FA Compounds EnzymeManipulation Organism SNF2 attenuate S. cerevisiae ¹⁶ IRA2 attenuate S.cerevisiae ¹⁶ PRE9 attenuate S. cerevisiae ¹⁶ PHO90 attenuate S.cerevisiae ¹⁶ SPT21 attenuate S. cerevisiae ¹⁶ ARE1 attenuate S.cerevisiae ¹⁷ ARE2 attenuate S. cerevisiae ¹⁷ DGA1 attenuate S.cerevisiae ¹⁷ LRO1 attenuate S. cerevisiae ¹⁷ ACL1 OE A. nidulans ALD6OE S. cerevisiae ACS1 OE S. enterica MAE1 OE S. cerevisiae GLC3attenuate S. cerevisiae GLG1, GLG2 attenuate S. cerevisiae

In general, cells do not naturally biosynthesize odd-chain alpha,α,ω-dicarboxylic acids. However, an odd-chain α,ω-dicarboxylic acid doesappear in biotin biosynthetic pathways usually as a bound intermediateto the acyl carrier protein of fatty acid biosynthesis. Biotinbiosynthesis is found in some, but not all organisms, which aretherefore auxotrophic for biotin. The bound intermediate is the C7α,ω-dicarboxylic acid, pimelic acid, which is always bound as apimeloyl-ACP until it is condensed with alanine where it is sequesteredinto the production of biotin. Until recently, the precise mechanism ofpimeloyl-ACP formation has remained elusive. Now E. coli's pathway tothis intermediate has been reported to proceed by methylatingmalonyl-CoA with a methyltransferase, BioC, followed by condensationwith malonyl-ACP to form 3-oxo-glutaryl-ACP methyl ester by fabH,followed by two full reduction-dehydration cycles and one extension bythe fatty acid synthases (fabG, fabZ, fabI, fabB). The methyl ester isthen converted into the free ω-carboxylic acid, pimeloyl-ACP by activityconferred by bioH (see Lin S, et al. Nature Chemical Biology 6, 682-688(2010)). A different pathway for biotin biosynthesis in B. subtilis hasbeen suggested to employ a P450 (BioI, CYP107H1) that in vitro isreported to cleave a carbon-carbon bond in a C14 fatty acyl-ACP to formtwo C7 molecules (see, e.g., Cryle, et al., “Structural insights from aP450 Carrier Protein complex reveal how specificity is achieved in theP450(BioI) ACP complex,” Proc Natl Acad Sci USA 105, 15696-701 (2008);and Cryle et al., “Products of cytochrome P450(BioI)(CYP107H1)-catalyzed oxidation of fatty acids,” Org Lett 5, 3341-4(2003), the teachings of both of which are incorporated by reference).Yet, this work remains unclear and no one has identified free pimelicacid from B. subtilis cultures, potentially due to the very low levelsof biotin required to support cell growth.

In accordance with the methods of this invention, we provide engineeredhost cells capable of producing odd-chain ω-hydroxyl fatty acids andα,ω-dicarboxylic acids in three general strategies that employ hybridType I PKSs, engineered portions of the B. subtilis biotin pathway, orengineered portions of the E. coli biotin pathway in a variety ofembodiments.

In one embodiment, a hybrid PKS system is used to produce heptanedioic(pimelic) acid, which is then oxidized with the methods described above.In one embodiment, a hybrid PKS is constructed and is composed of aloading module for either propionyl-CoA or methylmalonyl-CoA such thatthe starting unit is odd-chain. While there exist numerous loadingmodules that perform this function, one suitable loading module isselected from the Erythromycin PKS to load propionyl-CoA. This loadingmodule is operatively linked to two extension-condensation modules thatcondense malonyl-CoA into the growing acyl-ACP chain. One suitablechoice for these modules is the Nystatin PKS M5 and M15. Finally, thehybrid PKS is terminated with a thioesterase that cleaves the thioesterbond and releases heptanoic acid. One suitable choice for thethioesterase is that from the Erythromycin PKS (DEBS TE). This constructis cloned into an expression vector and transformed into cells that havephosphopantetheinylation activity to activate the hybrid PKS, the cellsare grown in appropriate medium and the free fatty acid heptanoic acidis produced. In some embodiments, the cells co-express the hybrid PKSand a set of enzymes for omega hydroxylation as described previously tofurther oxidize heptanoic acid into pimelic acid. In some embodimentsthe cells have no detectable levels of pimelic acid or do not have anyknown pathways for its production. In some embodiments the cells are C.tropicalis, B. subtilis, E. coli or S. cerevisiae.

In another embodiment, pimelic acid is produced by engineering a B.subtilis host. Specifically, engineered cells are provided thatoverexpress the gene encoding P450 BioI (bioI) and higher levels ofpimelic acid are detected (compared to wild-type). The enzyme cleavesthe central carbon-carbon bond in a C14 fatty acyl-ACP by consecutiveformation of alcohol and threo-diol intermediates to form pimelate. Inone embodiment, BioI is overexpressed by cloning it behind a regulatablepromoter for expression in B. subtilis and includes Pctc, PgsiB andresults in pimelic acid production. In other embodiments, BioI isoverexpressed by cloning it behind constitutive promoters derived fromthe sigma A or sigma B RNA polymerase promoter sequence. Additionally,biotin itself is a valuable chemical derived from pimelate, sooverproduction of pimelate in accordance with the methods and host cellsof the invention decreases the costs of microbial biotin production. Inanother embodiment, a thioesterase is expressed to increase the pimelateproduction. In another embodiment, the fatty acid synthesis enzymesnative to B. subtilis are overexpressed which results in increasedpimelate production. Suitable enzymes are provided in Table I.

In another embodiment, P450 bioI is overexpressed in E. coli, anon-native organism, with or without overexpression of the B. subtilisfatty acid enzymes (acpP, accABCD, KS, KR, ER, DH). In some embodiments,expression of a thioesterase is employed to increase release of thepimelate from the ACP. In another embodiment, the P450 BioI isoverexpressed in S. cerevisiae with or without the B. subtilis fattyacid enzymes (KS, KR, ER, DH, acpP) and a thioesterase and pimelate isproduced.

In another embodiment, fatty acid biosynthetic genes are expressed fromD. vulgaris or D. deslfuricans including the 3-oxoacyl acyl carrierprotein reductase (fabG) (Accession No. YP_(—)011773.1), the acpP(Accession No. YP_(—)011774.1), the beta ketoacyl ACP synthase (fabF)(Accession No. YP_(—)011775.1), the beta-hydroxyacyl ACP dehydratase(FabA/Z like) (Accession No. YP_(—)011772.1) and pimelate is produced.In some embodiments, additional expression of the NC_(—)002937 gene orthioesterase supports pimelate biosynthesis as an auxiliary proteinbased upon close proximity within the natural D. vulgaris gene cluster.

In another embodiment, E. coli genes are expressed in the native host ora heterologous host to produce pimeloyl-ACP and include bioC, fabF,fabG, fabA/Z, and fabI. In order to remove the methyl ester bonded tothe omega carboxylate, some embodiments include theexpression/overexpression of bioH. Once the omega carboxylate is exposeda fatty acid synthase editing TE releases the pimelic acid from theacp-thioester. This TE is naturally produced or can be overexpressed toincrease pimelic acid production. However, in some cases the pimelatemethyl ester is desired. In some embodiments, expression of athioesterase increases the production of pimelate or methyl pimelate. Inanother embodiment, the genes required for pimelic acid production in E.coli are expressed in S. cerevisiae and result in production of pimelicacid.

The enzymes described herein can be readily replaced using a homologousenzyme thereof A homologous enzyme is an enzyme that has a polypeptidesequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identicalto any one of the enzymes described in this specification or in anincorporated reference. The homologous enzyme retains amino acidsresidues that are recognized as conserved for the enzyme. The homologousenzyme may have non-conserved amino acid residues replaced or found tobe of a different amino acid, or amino acid(s) inserted or deleted, butwhich do not affect or has insignificant effect on the enzymaticactivity of the homologous enzyme. The homologous enzyme has anenzymatic activity that is identical or essentially identical to theenzymatic activity any one of the enzymes described in thisspecification or in an incorporated reference. The homologous enzyme maybe found in nature or be an engineered mutant thereof.

Nucleic acid constructs of the present invention comprise nucleic acidsequences encoding one or more of the subject enzymes. The nucleic acidof the subject enzymes are operably linked to promoters and optionallycontrol sequences such that the subject enzymes are expressed in a hostcell cultured under suitable conditions. The promoters and controlsequences are specific for each host cell species. In some embodiments,expression vectors comprise the nucleic acid constructs. Methods fordesigning and making nucleic acid constructs and expression vectors arewell known to those skilled in the art.

Sequences of nucleic acids encoding the subject enzymes are prepared byany suitable method known to those of ordinary skill in the art,including, for example, direct chemical synthesis or cloning. Further,nucleic acid sequences for use in the invention can be obtained fromcommercial vendors that provide de novo synthesis of the nucleic acids.

Each nucleic acid sequence encoding the desired subject enzyme can beincorporated into an expression vector. Incorporation of the individualnucleic acid sequences may be accomplished through known methods thatinclude, for example, the use of restriction enzymes (such as BamHI,EcoRI, HhaI, XhoI, XmaI, and so forth) to cleave specific sites in theexpression vector, e.g., plasmid. The restriction enzyme produces singlestranded ends that may be annealed to a nucleic acid sequence having, orsynthesized to have, a terminus with a sequence complementary to theends of the cleaved expression vector Annealing is performed using anappropriate enzyme, e.g., DNA ligase. As will be appreciated by those ofordinary skill in the art, both the expression vector and the desirednucleic acid sequence are often cleaved with the same restrictionenzyme, thereby assuring that the ends of the expression vector and theends of the nucleic acid sequence are complementary to each other. Inaddition, DNA linkers may be used to facilitate linking of nucleic acidssequences into an expression vector.

A series of individual nucleic acid sequences can also be combined byutilizing methods that are known to those having ordinary skill in theart (e.g., U.S. Pat. No. 4,683,195).

For example, each of the desired nucleic acid sequences can be initiallygenerated in a separate PCR. Thereafter, specific primers are designedsuch that the ends of the PCR products contain complementary sequences.When the PCR products are mixed, denatured, and reannealed, the strandshaving the matching sequences at their 3′ ends overlap and can act asprimers for each other Extension of this overlap by DNA polymeraseproduces a molecule in which the original sequences are “spliced”together. In this way, a series of individual nucleic acid sequences maybe “spliced” together and subsequently transduced into a host cellsimultaneously. Thus, expression of each of the plurality of nucleicacid sequences is effected.

Individual nucleic acid sequences, or “spliced” nucleic acid sequences,are then incorporated into an expression vector. The invention is notlimited with respect to the process by which the nucleic acid sequenceis incorporated into the expression vector. Those of ordinary skill inthe art are familiar with the necessary steps for incorporating anucleic acid sequence into an expression vector. A typical expressionvector contains the desired nucleic acid sequence preceded by one ormore regulatory regions, along with a ribosome binding site, e.g., anucleotide sequence that is 3-9 nucleotides in length and located 3-11nucleotides upstream of the initiation codon and followed by aterminator in the case of E. coli or other prokaryotic hosts. See Shineet al. (1975) Nature 254:34 and Steitz, in Biological Regulation andDevelopment: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349,1979, Plenum Publishing, N.Y. In the case of eukaryotic hosts like yeasta typical expression vector contains the desired nucleic acid sequencepreceded by one or more regulatory regions, along with a Kozak sequenceto initiate translation and followed by a terminator. See Kozak M(1984). Nature 308 (5956): 241-246.

Regulatory regions include, for example, those regions that contain apromoter and an operator. A promoter is operably linked to the desirednucleic acid sequence, thereby initiating transcription of the nucleicacid sequence via an RNA polymerase enzyme. An operator is a sequence ofnucleic acids adjacent to the promoter, which contains a protein-bindingdomain where a repressor protein can bind. In the absence of a repressorprotein, transcription initiates through the promoter. When present, therepressor protein specific to the protein-binding domain of the operatorbinds to the operator, thereby inhibiting transcription. In this way,control of transcription is accomplished, based upon the particularregulatory regions used and the presence or absence of the correspondingrepressor protein. Examples for prokaryotic expression include lactosepromoters (LacI repressor protein changes conformation when contactedwith lactose, thereby preventing the LacI repressor protein from bindingto the operator) and tryptophan promoters (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator). Another example is thetac promoter. (See deBoer et al. (1983) Proc. Natl. Acad. Sci. USA,80:21-25.). Examples of promoters to use for eukaryotic expressioninclude pTDH3, pTEF1, pTEF2, pRNR2, pRPL18B, pREV1, pGAL1, pGAL10,pGAPDH, pCUP1, pMET3, pPGK1, pPYK1, pHXT7, pPDC1, pFBA1, pTDH2, pPGI1,pPDC1, pTPI1, pENO₂, pADH1, and pADH2. As will be appreciated by thoseof ordinary skill in the art, these and other expression vectors orelements may be used in the present invention, and the invention is notlimited in this respect.

Although any suitable expression vector may be used to incorporate thedesired sequences, readily available expression vectors include, withoutlimitation: plasmids, such as pSC101, pBR322, pBBR1MCS-3, pUR, pEX,pMR100, pCR4, pBAD24, pUC19, pRS series; bacteriophages, such as M13phage and λ phage. Of course, such expression vectors may only besuitable for particular host cells. One of ordinary skill in the art,however, can readily determine through routine experimentation whetherany particular expression vector is suited for any given host cell. Forexample, the expression vector can be introduced into the host cell,which is then monitored for viability and expression of the sequencescontained in the vector. In addition, reference may be made to therelevant texts and literature, which describe expression vectors andtheir suitability to any particular host cell. In addition to the use ofexpression vectors, strains are built where expression cassettes aredirectly integrated into the host genome.

The expression vectors or integration cassettes of the invention must beintroduced or transferred into the host cell. Such methods fortransferring the expression vectors into host cells are well known tothose of ordinary skill in the art. For example, one method fortransforming E. coli with an expression vector involves a calciumchloride treatment wherein the expression vector is introduced via acalcium precipitate. Other salts, e.g., calcium phosphate, may also beused following a similar procedure. In addition, electroporation (i.e.,the application of current to increase the permeability of cells tonucleic acid sequences) may be used to transfect the host microorganism.Also, microinjection of the nucleic acid sequencers) provides theability to transfect host microorganisms. Other means, such as lipidcomplexes, liposomes, and dendrimers, may also be employed. Those ofordinary skill in the art can transfect a host cell with a desiredsequence using these or other methods.

For identifying a transfected host cell, a variety of methods areavailable. For example, a culture of potentially transfected host cellsmay be separated, using a suitable dilution, into individual cells andthereafter individually grown and tested for expression of the desirednucleic acid sequence. In addition, when plasmids are used, anoften-used practice involves the selection of cells based uponantimicrobial resistance that has been conferred by genes intentionallycontained within the expression vector, such as the amp, gpt, neo, andhyg genes.

The host cell is transformed with at least one expression vector. Whenonly a single expression vector is used (without the addition of anintermediate), the vector will contain all of the nucleic acid sequencesnecessary.

Once the host cell has been transformed with the expression vector, thehost cell is allowed to grow. For microbial hosts, this process entailsculturing the cells in a suitable medium. It is important that theculture medium contain a carbon source, such as a sugar (e.g., glucose)when an intermediate is not introduced. In this way, cellular productionof acetyl-CoA, the starting material for the production of the diacids,is ensured. When added, the intermediate is present in an excess amountin the culture medium or cells.

As the host cell grows and/or multiplies, expression of the enzymesnecessary for producing the fatty acid, hydroxyl fatty acid, 1-oxo fattyacid, 1-ol fatty acid and the diacid is effected. Once expressed, theenzymes catalyze the steps necessary for carrying out the enzymaticsteps shown in FIGS. 1 and 4. If an intermediate has been introduced,the expressed enzymes catalyze those steps necessary to convert theintermediate into the respective fatty acid derived compounds. Any meansfor recovering the diacid from the host cell may be used. For example,the host cell may be harvested and subjected to hypotonic conditions,thereby lysing the cells. The lysate may then be centrifuged and thesupernatant subjected to high performance liquid chromatography (HPLC)or gas chromatography (GC).

Host Cells

The host cells of the present invention are genetically modified in thatheterologous nucleic acid have been introduced into the host cells ornaturally occurring cells have been engineered to produce higher levelsof a given product, and as such the genetically modified host cells donot occur in nature. The suitable host cell is one capable of expressinga nucleic acid construct encoding an enzyme capable of catalyzing adesired biosynthetic reaction in order to produce the enzyme forproducing the desired fatty acid or fatty acid derived molecule. Suchenzymes are described herein. In some embodiments, the host cellnaturally produces some of the precursors, as shown in FIGS. 1 and 4,for the production of the fatty acid derived compounds. These genesencoding the desired enzymes may be heterologous to the host cell orthese genes may be native to the host cell but are operatively linked toheterologous promoters and/or control regions, which result in thehigher expression of the gene(s) in the host cell. In other embodiments,the host cell does not naturally produce the fatty acid startingmaterial and comprises heterologous nucleic acid constructs capable ofexpressing one or more genes necessary for producing the fatty acid.

Each of the desired enzymes capable of catalyzing the desired reactioncan be native or heterologous to the host cell. Where the enzyme isnative to the host cell, the host cell is optionally geneticallymodified to modulate expression of the enzyme. This modification caninvolve the modification of the chromosomal gene encoding the enzyme inthe host cell or a nucleic acid construct encoding the gene of theenzyme is introduced into the host cell. One of the effects of themodification is the expression of the enzyme is modulated in the hostcell, such as the increased expression of the enzyme in the host cell ascompared to the expression of the enzyme in an unmodified host cell.

The genetically modified host cell can further comprise a geneticmodification whereby the host cell is modified by the increasedexpression of one or more genes involved in the production of fatty acidcompounds from one of five methods provided such that the production offatty acid compounds by the host cell is increased. Such genes encodeenzymes related to either Type I, II, or III fatty acid biosynthesis,hybrid Type I polyketide synthesis, or 2-ketoacid biosynthesis andinclude: acetyl carboxylase (ACC), ketosynthase, ketoreductase,deyhdratase, enoyl reductase, cytosolic thiosterase (‘TesA, sometimesreferred to as LTesA), and acyl-carrier protein (AcpP). In someembodiments, the genetically modified host cell is modified to producehigher levels of cytosolic acetyl-coA or malonyl-CoA or the pathway maybe targeted to the mitochondria or compartment where there is a naturalor engineered abundance of acetyl-CoA and other necessary precursors.Thus, in some embodiments, a host cell of the invention comprises amodification to express, or increase expression of a protein such as ATPcitrate lyase, and to increase levels of NADPH, malic enzyme. Forexample, Saccharomyces cerevisiae has little ATP citrate lyase and canbe engineered in accordance with the invention to express ATP citratelyase by introducing an expression vector encoding ATP citrate lyaseinto the yeast cells.

In some embodiments, a genetically modified host cell is modified toincrease expression of a Type I (prokaryotic, eukaryotic) or Type II(prokaryotic) or Type III fatty acid synthase (FAS) gene or Type Ipolyketide synthase (PKS) or 2-ketoacid biosynthetic enzymes. Forexample, a yeast host cell is modified to express a FAS gene. Fatty acidsynthase proteins are known in the art. FAS3 catalyzes the firstcommitted step in fatty acid biosynthesis and in yeast is encoded by a6.7 kb gene and contains two enzymatic domains: biotin carboxylase, andbiotin carboxyltransferase. FAS2 is encoded, in yeast, by a 5.7 kb geneand contains four domains: an acyl-carrier protein, beta-ketoacylreductase, beta-ketoacyl synthase, and phosphopantetheinyl transferase(PPT). FAS 1 is encoded, in yeast, by a 6.2 kb gene and contains fivedomains: acetyltransacylase, dehydratase, enoyl reductase, malonyltransacylase, and palmitoyl transacylase. FAS 1 and FAS2 complex to forma heterododecamer, containing six each of FAS1 and FAS2 subunits(Lomakin et al., Cell 129:319-322, 2007, incorporated herein byreference). In some embodiments, a genetically modified host celloverexpresses or expresses native and/or non-native type II fatty acidsynthase enzymes. Illustrative genes that encode the enzymes areprovided: acpP, accA, accB, accC, accD, fabD, fabH, fabG, fabZ, fabA,fabI, fabB, fabF, fadR. In some embodiments a genetically modified hostcell overexpresses or expresses hybrid engineered type I polyketidesynthases.

The genetically modified host cell can further comprise a geneticmodification whereby the host cell is modified by the decreased or lackof expression of one or more genes encoding proteins involved in thestorage and/or metabolism of fatty acid compounds; such that the storageand/or metabolism of fatty acid compounds by the host cell is decreased.Such genes include the following: the ARE1, ARE2, DGA1, and/or LRO1genes. In some embodiments, the host cell is modified by the decreasedor lack of expression of genes that are involved in the β-oxidation offatty acids. For example, in yeast such, e.g., Saccharomyces cerevisiae,β-oxidation occurs in the peroxisome. Genes such as PAT1 and PEX11 areperoxisomal proteins involved in degradation of long-chain andmedium-chain fatty acids, respectively. Accordingly, a host cell may bemodified in accordance with the invention to delete PAT1 and/or PEX11,or otherwise decrease expression of the PAT1 and/or PEX11 proteins.

The genetically modified host cell of the invention can further comprisea genetic modification whereby the host cell is modified to express orhave increased expression of an ABC transporter that is capable ofexporting or increasing the export of any of the fatty acid derivedcompounds from the host cell. Such an ABC transporter is the plant cer5or dcuC.

The present invention provides a wide variety of prokaryotic oreukaryotic host cell suitable for use in the present method and methodsfor making such host cells. In some embodiments, the bacteria is acyanobacteria. Examples of suitable bacterial host cells include,without limitation, those species assigned to the Escherichia,Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia,Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla,Synechococcus, Synechocystis, and Paracoccus taxonomical classes.

Suitable eukaryotic cells include, but are not limited to, fungal,insect or mammalian cells. Suitable fungal cells are yeast cells, suchas yeast cells of the Saccharomyces genus. In some embodiments theeukaryotic cell is an algae, e.g., Chlamydomonas reinhardtii,Scenedesmus obliquus, Chlorella vulgaris or Dunaliella salina.

In some embodiments, the host organism is yeast. Suitable yeast hostcells include, but are not limited to, Yarrowia, Candida, Bebaromyces,Saccharomyces, Schizosaccharomyces and Pichia. In one embodiment, theyeast host cell is a species of Candida selected from the groupconsisting of C. tropicalis, C. maltosa, C. apicola, C. paratropicalis,C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica,C. panapsilosis and C. zeylenoides. In one embodiment, Candidatropicalis is employed as the host organism.

The present invention provides for an isolated fatty acid derivedcompound produced by the method of the present invention. Isolating thefatty acid derived compound involves the separating at least part or allof the fermentation medium, host cells, and parts thereof, from whichthe fatty acid derived compound was produced, from the isolated fattyacid derived compound. The isolated fatty acid derived compound may befree or essentially free of impurities formed from at least part or allof the host cells, and parts thereof. The isolated fatty acid derivedcompound is essentially free of these impurities when the amount andproperties of the impurities do not interfere in the subsequent use ofthe fatty acid derived compound. For example, if the subsequent use isas an industrial chemical, such as a chemical to be used in apolymerization reaction, then the compound is essentially free ofimpurities when any remaining impurities would not interfere with theuse of the compound as an industrial chemical in a polymerizationreaction or any other downstream industrial reaction. If the product isto be used as a fuel, such as a fuel to be used in a combustionreaction, then the compound is essentially free of impurities when anyimpurities remaining would not interfere with the use of the material asa fuel. In some instances, the host cells of the invention do notnaturally produce the desired fatty acid derived compound.

The fatty acid derived compound of the present invention are useful notonly as fuels as a chemical source of energy but also as industrialchemicals and precursors thereof that can be used as an alternative topetroleum derived fuels, ethanol and the like, and industrial chemicalsand their precursors. The fatty acid derived compounds of the presentinvention are also useful in the synthesis of alkanes, alcohols, andesters of various for use as a renewable fuel or for industrial chemicalproduction. In addition, the fatty acid derived compounds can also be asprecursors in the synthesis of therapeutics, high-value oils, such as acocoa butter equivalent and animal feeds. The fatty acid derivedcompounds are also useful in the production of the class of eicosanoidsor related molecules, which have therapeutic related applications.

It is to be understood that, while the invention is described herein inconjunction with specific embodiments thereof, the foregoing descriptionis intended to illustrate and not limit the scope of the invention.Other aspects, advantages, and modifications within the scope of theinvention will be apparent to those skilled in the art to which theinvention pertains in view of this disclosure.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

The invention having been described, the following examples are offeredto illustrate the subject invention by way of illustration, not by wayof limitation.

EXAMPLES Example 1 Production of Tetradecanedioic Acid

In accordance with various embodiments of the present invention,tetradecanedioic acid is produced. In one embodiment, a heterologoustype III fatty acid synthase ELO1 and ELO2 (T. brucei) are expressed inconjunction with a set of enzymes that encode for the production ofbutyryl-CoA, which in some embodiments include phaA, phaB, phaJ, ter,and a thioesterase, which in some embodiments are encoded by ltesA toproduce tetradecanoic acid. The tetradecanoic acid is then convertedthrough the alcohol and aldehyde intermediate into the diacidtetradecanedioic acid by expressing a P450 and FAO.

In another embodiment, a native type II FAS system and a thioesterasefrom E. coli that produces tetradecanoate were expressed in a host cellof the invention. Once produced, the tetradecanoate is further oxidizedin accordance with the invention at the omega carbon by P450s. Althoughmany thioesterases are suitable for these embodiments, in one embodimentthe 1TesA from E. coli is employed. Although many P450s or omegaoxidases are suitable for these embodiments, in one embodiment the P450BM3 from B. subtilis is employed. Briefly, P450 BM3 and a mutant P450BM3 (F87A) were separately cloned using standard methods and insertedbehind the TAC promoter on an E. coli expression plasmid that containedthe p15a origin of replication, and an ampicillin resistant gene. Athioesterase, 1TesA was cloned using standard methods and insertedbehind the LacUV5 promoter on an E. coli expression plasmid thatcontained the pBBR origin of replication and a tetracycline resistancegene. Plasmid maps are included in FIG. 3. The host cells harboringexpression plasmids for the LtesA and p450 BM3 or LtesA and p450 BM3(F87A) were grown in LB media at 37 degrees C. and induced at OD=0.5with 1 mM IPTG. Cells were grown for 48 h and separated from thesupernatant by centrifugation. Both supernatant and pellet fraction wereseparately dried and resuspended in MeOH:H20 (1:1 v/v). Chemicalstandards were purchased from Sigma and were made up to 20 μM, inmethanol and water (1:1, v/v). The separation of diacids was conductedon a ZIC-HILIC column (250 mm length, 2.1 mm internal diameter, and 3.5μm particle size; from Merck SeQuant, and distributed via The NestGroup, Inc., MA., USA) using an Agilent Technologies 1200 Series HPLCsystem (Agilent Technologies, CA, USA). An injection volume of 4 μL wasused throughout. The auto-sample tray was maintained at 4° C. by anAgilent FC/ALS Thermostat. The column compartment was set to 50° C.Analytes were eluted isocratically with a mobile phase composition of 50mM ammonium acetate, in water, and acetonitrile (3.6:6.4, v/v). A flowrate of 0.1 mL/min was used throughout.

The HPLC system was coupled to an Agilent Technologies 6210time-of-flight mass spectrometer (LC-TOF MS), by a ⅓ post-column split.Contact between both instrument set-ups was established by a LAN card totrigger the MS into operation upon the initiation of a run cycle fromthe MassHunter workstation (Agilent Technologies, CA, USA). Electrosprayionization (ESI) was conducted in the negative ion mode and a capillaryvoltage of −3500 V was utilized. MS experiments were carried out in fullscan mode, at 0.85 spectra/second and a cycle time of 1.176 seconds, forthe detection of [M-H]-ions. The instrument was tuned for a range of50-1700 m/z. Prior to LC-TOF MS analysis, the TOF MS was calibrated viaan ESI-L-low concentration tuning mix (Agilent Technologies, CA, USA).Data acquisition and processing were performed by the MassHuntersoftware package (Agilent Technologies, CA, USA).

FIGS. 2 and 3 illustrate that the expression of both the wild-type P450Bm3 and the engineered P450 BM3 (F87A) resulted in diacid productionwhen the native E. coli fatty acid pathway is overexpressed via LtesA.Data shown in FIG. 2 demonstrated production and excretion of the C14α,ω-dicarboxylic acid tetradecanoate. Specifically, FIG. 2A demonstratedproduction of both the C14 ω-hydroxytetradecanoic acid andtetradecanedioic acid. FIG. 2B is MS data that show the expectedmolecular ion of tetradecanedioic acid.

Example 2 Production of Butanedioic Acid

In accordance with the methods of the invention, butanedioic is producedby a variety of embodiments. This example describes convertingacetyl-CoA into butyryl-CoA, which is cleaved from the CoA by athioesterase (TES), oxidized by a P450 monooxygenase (OX1) and furtheroxidized into its respective diacid by another oxidase (OX2).Importantly, a thioesterase can be engineered in accordance with theinvention so that it cleaves hydroxybutyrate from its CoA, allowing forthe production of 2-hydroxybutanedioic acid.

The above method is merely illustrative of the many embodiments providedby the invention. Another embodiment involves the use of a PKS composedof a loading module that incorporates malonyl-CoA an extension moduleand a TE that releases a butyrate product. In this embodiment, the fattyacid is then oxidized into omega hydroxybutyrate and finally intobutanedioic acid. While many PKS modules can be used, in one embodiment,a malonyl-CoA loading module from the niddamycin PKS is functionallyattached to an extension and full reduction module from the nystatin PKS(module 5) to provide a PKS of the invention. To release the product asa fatty acid, a TE, for example, the DEBS PKS TE, is functionallyattached to the nystatin module. This sequence is placed into anexpression vector for E. coli, expressed at 15C-37 degrees C. andresults in production of butyric acid (SEQ ID NO:1 below). Additionalexpression of a short chain oxidase (OX1 and OX2) further results inproduction of butanedioic acid in accordance with the invention.

Example 3 Production of Hexanedioic Acid

In accordance with various embodiments of the methods of the invention,hexanedioic acid is produced. In one embodiment, genes in the aflatoxinbiosynthesis pathway that encode fatty acid biosynthesis reactionsresulting in a final C6 fatty acid product, hexAB are used. Becauseothers have reported that the hexanoic acid is bound to the hexABenzyme³, in some embodiments, the hexAB has been mutated to decrease oreliminate such binding, and in other embodiments, a thioesterase (TES)is expressed that cleaves the C6 fatty acid from its thioester. Inanother embodiment, the transacylase from the PKS is used to load an ACPand subsequently cleave it with a TES. In another alternativeembodiment, a short-chain thioesterase is engineered to directly producethe C6 fatty acid from type II fatty acid biosynthesis (see, e.g,Dehesh, et al., “Production of high levels of 8:0 and 10:0 fatty acidsin transgenic canola by overexpression of Ch FatB2, a thioesterase cDNAfrom Cuphea hookeriana.” Plant J 9, 167-72 (1996)). Once produced, thehexanoate is further oxidized in accordance with the invention at theomega carbon by P450s. Although many P450s or omega oxidases aresuitable for these embodiments, in one embodiment the P450 BM3 from B.subtilis is employed, either native or engineered. In variousembodiments, additional expression of a P450 monoxygenase and reductase(P4503P2 and CPR) further results in production of adipate, inaccordance with the invention.

Scheme II

The above embodiments are merely illustrative. Another embodimentutilizes an engineered PKS of the invention composed of a loading modulethat incorporates malonyl-CoA, two extension modules, and a TE thatreleases a final hexanoate product. This fatty acid is then oxidizedinto omega hydroxybutyrate and finally into hexanedioic acid (adipate)by, in one embodiment, expression of P4503P2, CPR, and a FAO/ALD in thehost cell. More specifically, a malonyl-CoA loading module from theniddamycin PKS is functionally attached to an extension and fullreduction module from the nystatin PKS (module 5), followed by anotherextension and full reduction module from the nystatin PKS (module 15) toprovide a PKS of the invention. To release the product as a fatty acid,a TE, optionally the DEBS PKS TE, is functionally attached to the lastnystatin module. This sequence is placed into an expression vector forE. coli, expressed at 15-37 degrees C., resulting in production ofhexanoic acid (sequence hex orf 1 & hex orf 2, i.e, SEQ ID NOS:2 and 3,respectively, below). Additional expression of a short chain oxidase(OX1 and OX2) in accordance with the invention further results inproduction of adipic acid.

Example 4 Production of Octanedioic Acid

In accordance with various embodiments of the present invention,octanedioic acid is produced. In one embodiment, engineered host cellsare provided that express a native type II FAS system and a heterologousthioesterase, ChFatB2 from C. hookeriana to produce octanoate. Theoctanoate is further converted into the diacid octanedioic acid byexpression of a hydroxylase (P4503P2) and an FAO/FAD, ADH combination.

Scheme III Example 5 Production of Decanedioic Acid

In accordance with various embodiments of the present invention,decanedioic acid is produced. The thioesterase, ChFatB2, from C.hookeriana, produces decanoate. In one embodiment, host cells aremodified to produce decanedioic acid by expressing a native type II FASsystem and a thioesterase, ChFatB2 from C. hookeriana, to producedecanoate. In another embodiment host cells are modified to producedecanedioic acid by expressing a type III fatty acid synthase, ELO1 (T.brucei) and has appropriate genes for the production of butyryl-CoA,which in some embodiments include phaA, phaB, phaJ, ter, and athioesterase, which in some embodiments are encoded by ChFatB2. Althoughmany P450s could be employed, in one embodiment, the decanoate is thenconverted in accordance with the invention into the diacid decanedioicacid by expressing P4503P2 and FAO/FAD, ADH combination.

Scheme IV Example 6 Production of Dodecanedioic Acid

In accordance with various embodiments of the present invention,dodecanedioic acid is produced. In one embodiment, a host cellcontaining a native type II FAS system and a heterologous thioesterase,UcFATB1 from U. californica that produces dodecanoate, is provided. Thedodecanoate can be further oxidized in accordance with the invention atthe omega carbon by P450s.

Scheme V Example 7 Production of C14(n+2) α,ω-Dicarboxylic Acids

The methods presented above can be used to produce of longer chaindiacids up to C26 and longer, with longer chain fatty acid biosynthesissystems existing in organisms such as mycoplasms, etc.

Example 8 Engineering Thioesterase Substrate Specificity

Thioesterase substrate specificity can be engineered in accordance withthe methods of the invention to produce specific fatty acid chainlengths (see, e.g., Yuan et al., Proc Natl Acad Sci USA 92, 10639-43(1995); see also, references 4 and 6).

Example 9 Oxidation by P450 BM3

Terminal oxidation can be carried out by the wild-type P450 BM3monoxygenase using an ω-hydroxyfatty acid as a substrate (see, e.g.,Schneider et al., “Production of alkanedioic acids by cytochrome P450BM3 monooxygenase: oxidation of 16-hydroxyhexadecanoic acid tohexadecane-1,16-dioic acid,” Biocataysis and Biotransformation,17:163-178 (1999)). Thus, in addition to the previous examples thatutilize fatty alcohol oxidation/fatty alcohol dehydration and aldehydedehydration, the final oxidation is, in some embodiments, carried out bythe P450 BM3 enzyme. Further, the hydroxylation position can be changedto the {tilde over (ω)}carbon by a point mutation, resulting inω-hydroxylation of laurate (see, Oliver, et al., “A single mutation incytochrome P450 BM3 changes substrate orientation in a catalyticintermediate and the regiospecificity of hydroxylation,” Biochemistry36, 1567-72 (1997)). Thus, expression of both the wild-type P450 BM3 andthe engineered P450 BM3 (F87A) results in diacid production if fattyacids are supplied. Further, the substrate specificity can be changed toshorter chain length fatty acids by introduction of various pointmutations, resulting in oxidation of short chain length substrates (see,Ost, et al. “Rational re-design of the substrate binding site offlavocytochrome P450 BM3.” FEBS Letters 486, 173-177 (2000)).

Example 10 Controlling Saturation

Fatty acid saturation can be controlled by expressing desaturases or,alternatively, by overexpressing fadR.

Example 11 Controlling Internal Hydroxylation

Other P450s hydroxylate various ω-1,2,3 positions and produce long chainmolecules that resemble polyhydroxyalkanoates. Alternatively, one cancleave the thioester early in the fatty acid reduction/elongation cycleto produce molecules like 2-hydroxymyristate in accordance withembodiments of the invention.

Example 12 Biosynthetic Route to Omega Hydroxy Fatty Acids

Omega hydroxy fatty acids themselves are valuable as polymer substratesand can easily be produced with an embodiment of the invention in whichexample number 6 above is utilized after eliminating the FAO/FAD and ADHenzyme activities.

Example 13 Providing Fatty Acid Substrate Through Type I Fatty AcidBiosynthesis

Alternate methods of the invention utilize Type I fatty acidbiosynthesis for controlling fatty acid chain length through short chainelongation systems. This results in production of specific acyl-CoAchain lengths ranging from C4, C10, C14, C18, C20, C22, and C26. Thefatty acid substrates are cleaved from the CoA thioester by expressing athioesterase that has broad substrate range. The fatty acid of desiredchain length can then be omega oxidized to form its respective diacid,as described in Examples 1-6.

Example 14 Production of Odd Chain α,ω-Dicarboxylic Acids

Odd chain diacids are also valuable molecules that can be producedthrough decarbonylation of fatty acids to produce an odd chain fattyacids in accordance with the invention. This odd chain fatty acids arethen oxidized utilizing the oxidation methods described herein.Alternatively, odd chain fatty acids are produced when propionyl-CoA isused as a primer for fatty acid or polyketide synthases (instead ofacetyl-CoA). Once the odd chain fatty acids are produced via thesemethods, they proceed through omega oxidation as described above.

Example 15 Production of C7 Diacid (Pimelic Acid)

Pimelic acid is a precursor to the biotin biosynthesis pathway and isproduced naturally in different organisms by different pathways relatingto fatty acid like mechanisms. The present invention provides a varietyof embodiments for the production of pimelic acid.

In one embodiment, the gene encoding native or engineered P450 BioI(bioI) native to B. subtilis is overexpressed in B. subtilis by cloningbehind a sigma B RNA polymerase constitutive promoter and higher levelsof pimelic acid are detected (compared to wild-type). The enzyme cleavesthe central carbon-carbon bond in a C14 fatty acyl-ACP by consecutiveformation of alcohol and threo-diol intermediates to form pimelate.Additionally, biotin itself is a valuable chemical derived frompimelate, so overproduction of pimelate in accordance with the methodsand host cells of the invention decreases the costs of microbial biotinproduction.

In another embodiment, P450 bioI is overexpressed in E. coli, anon-native organism, with or without overexpression of the B. subtilisfatty acid enzymes (acpP, accABCD, KS, KR, ER, DH). In some embodiments,expression of a thioesterase is employed to increase release of thepimelate from the ACP.

In another embodiment, the P450 BioI is overexpressed in S. cerevisiaewith or without the B. subtilis fatty acid enzymes (KS, KR, ER, DH).

In another embodiment, the orfs following bioI, ytbQ and ytcP and ytcQ(B. subtilis) are expressed to increase pimelate production. In anotherembodiment, a thioesterase is expressed to increase the pimelateproduction.

In another embodiment, a hybrid PKS system is used to produceheptanedioic acid which is then oxidized with the methods describedabove. Specifically, a PKS propionyl-CoA or methylmalonyl-CoA isfunctionally linked to two malonyl-CoA extension and reduction modulesand finally to a TE module and expressed in E. coli to produce heptanoicacid. Expression of the omega oxidizing enzymes results in oxidation ofthe heptanoic acid to heptanedioic (pimelic) acid.

In another embodiment, fatty acid biosynthetic genes are expressed fromD. vulgaris or D. deslfuricans including the 3-oxoacyl acyl carrierprotein reductase (fabG) (Accession No. YP_(—)011773.1), the acpP(Accession No. YP_(—)011774.1), the beta ketoacyl ACP synthase (fabF)(Accession No. YP_(—)011775.1), the beta-hydroxyacyl ACP dehydratase(FabA/Z like) (Accession No. YP_(—)011772.1) to produce pimelate. Insome embodiments, additional expression of the NC_(—)002937 gene orthioesterase supports pimelate biosynthesis as an auxiliary proteinbased upon close proximity within the natural D. vulgaris gene cluster.

In another embodiment, E. coli genes are expressed in the native host ora heterologous host to produce pimeloyl-ACP and include bioC, fabF,fabG, fabA/Z, and fabI. In order to remove the methyl ester bonded tothe omega carboxylate, some embodiments include theexpression/overexpression of bioH, however in some cases, the pimelatemethyl ester is desired. In some embodiments, expression of athioesterase increases the production of pimelate or methyl pimelate.

LISTING OF REFERENCES

-   1. Mobley, D. Biosynthesis of long-chain dicarboxylic acid monomers    from renewable resources. US Department of Energy Report (1999).-   2. Picataggio, S. et al. Metabolic engineering of Candida tropicalis    for the production of long-chain dicarboxylic acids. Biotechnology    (N Y) 10, 894-8 (1992).-   3. Watanabe, C. M. & Townsend, C. A. Initial characterization of a    type I fatty acid synthase and polyketide synthase multienzyme    complex N or S in the biosynthesis of aflatoxin B(1). Chem Biol 9,    981-8 (2002).-   4. Yuan, L., Voelker, T. A. & Hawkins, D. J. Modification of the    substrate specificity of an acyl-acyl carrier protein thioesterase    by protein engineering. Proc Natl Acad Sci USA 92, 10639-43 (1995).-   5. Dehesh, K., Jones, A., Knutzon, D. S. & Voelker, T. A. Production    of high levels of 8:0 and 10:0 fatty acids in transgenic canola by    overexpression of Ch FatB2, a thioesterase cDNA from Cuphea    hookeriana. Plant J 9, 167-72 (1996).-   6. Yuan, L. (Calgene, Dec. 14, 2005).-   7. S Schneider, M. W., D Sanglard, B Witholt. Production of    alkanedioic acids by cytochrome P450 BM-3 monooxygenase: oxidation    of 16-hydroxyhexadecanoic acid to hexadecane-1,16-dioic acid.    Biocataysis and biotransformation 17, 163-178 (1999).-   8. Oliver, C. F. et al. A single mutation in cytochrome P450 BM3    changes substrate orientation in a catalytic intermediate and the    regiospecificity of hydroxylation. Biochemistry 36, 1567-72 (1997).-   9. Craft, D. L., Madduri, K. M., Eshoo, M. & Wilson, C. R.    Identification and characterization of the CYP52 family of Candida    tropicalis ATCC 20336, important for the conversion of fatty acids    and alkanes to alpha,omega-dicarboxylic acids. Appl Environ    Microbiol 69, 5983-91 (2003).-   10. Seghezzi, W. et al. Identification and characterization of    additional members of the cytochrome P450 multigene family CYP52 of    Candida tropicalis. DNA Cell Biol 11, 767-80 (1992).-   11. Imai, Y. Characterization of rabbit liver cytochrome P-450    (laurate omega-1 hydroxylase) synthesized in transformed yeast    cells. J Biochem 103, 143-8 (1988).-   12. Hardwick, J. P. Cytochrome P450 omega hydroxylase (CYP4)    function in fatty acid metabolism and metabolic diseases. Biochem    Pharmacol 75, 2263-75 (2008).-   13. Lee, S. H., Stephens, J. L., Paul, K. S. & Englund, P. T. Fatty    acid synthesis by elongases in trypanosomes. Cell 126, 691-9 (2006).-   14. Erdmann, R., Veenhuis, M., Mertens, D. & Kunau, W. H. Isolation    of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc    Natl Acad Sci USA 86, 5419-23 (1989).-   15. Scharnewski, M., Pongdontri, P., Mora, G., Hoppert, M. &    Fulda, M. Mutants of Saccharomyces cerevisiae deficient in acyl-CoA    synthetases secrete fatty acids due to interrupted fatty acid    recycling. Febs J 275, 2765-78 (2008).-   16. Kamisaka, Y. et al. Identification of genes affecting lipid    content using transposon mutagenesis in Saccharomyces cerevisiae.    Biosci Biotechnol Biochem 70, 646-53 (2006).-   17. Sandager, L. et al. Storage lipid synthesis is non-essential in    yeast. J Biol Chem 277, 6478-82 (2002).-   18. Cryle, M. J. & Schlichting, I. Structural insights from a P450    Carrier Protein complex reveal how specificity is achieved in the    P450(BioI) ACP complex. Proc Natl Acad Sci U S A 105, 15696-701    (2008).-   19. Cryle, M. J., Matovic, N. J. & De Voss, J. J. Products of    cytochrome P450(BioI) (CYP107H1)-catalyzed oxidation of fatty acids.    Org Lett 5, 3341-4 (2003).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A recombinant cell that produces anomega-hydroxylated fatty acid or a dicarboxyic acid or both fromendogenous fatty acids, wherein said cell comprises (i) a Type I fattyacid biosynthesis pathway and a fatty acid omega hydroxylase; (ii) aType II fatty acid biosynthesis pathway and a fatty acid omegahydroxylase; (iii) a Type III fatty acid biosynthesis pathway and afatty acid omega hydroxylase; (iv) a Type I polyketide synthase (PKS)pathway and a fatty acid omega hydroxylase; (v) a 2-keto acidbiosynthesis pathway and a fatty acid omega hydroxylase; and (vi) abiotin biosynthesis pathway and a cytochrome P450 oxidase; wherein oneor more of said fatty acid omega hydroxylase, cytochrome P450 oxidase,and biosynthesis pathway enzymes is encoded by a recombinant nucleicacid in said cell.
 2. The recombinant cell of claim 1, wherein at leasttwo of said fatty acid omega hydroxylase, cytochrome P450 oxidase, andbiosynthesis pathway enzymes is encoded by a recombinant nucleic acid insaid cell.
 3. The recombinant cell of claim 1, wherein said fatty acidis produced by a Type 1, II, or III fatty acid biosynthesis pathway andsaid fatty acid omega hydroxylase is encoded by a recombinant nucleicacid in said cell.
 4. The recombinant cell of claim 1, wherein saidfatty acid is produced by a Type I PKS pathway and said Type I PKS isencoded by a recombinant nucleic acid in said cell.
 5. The recombinanthost cell of claim 1, wherein said fatty acid is produced by a 2-ketoacid biosynthesis pathway that includes mutated LeuA and KIVD encoded bya recombinant nucleic acid in said cell.
 6. The recombinant host cell ofclaim 1, wherein said fatty acid is produced by a biotin biosynthesispathway, at least one enzyme of which is encoded by a recombinantnucleic acid in said cell, and said cell produces pimelic acid.
 7. Therecombinant cell of claim 1 that produces an alpha, omega-dicarboxylicacid by conversion of said omega-hydroxylated fatty acid with a fattyacid oxidase and aldehyde dehydrogenase enzymes.
 8. The recombinant cellof claim 1, wherein the fatty acid omega hydroxylase is selected fromthe group consisting of P450 (3P2), P450 (PHP3), and P450 BM3 (F87A). 9.The recombinant cell of claim 1, wherein the host cell has beengenetically modified to reduce β-oxidation.
 10. The recombinant cell ofclaim 6, wherein the dicarboxylic acid has chain length from C3 to C26.11. The recombinant cell of claim 1, wherein the cell further comprisesa genetic modification selected from the group consisting of (i) agenetic modification that increases the expression of one or more genesinvolved in the production of fatty acid compounds is increased; (ii) agenetic modification that decreases the expression of one or more genesencoding proteins involved in the storage or metabolism of fatty acidcompounds; and (iii) a genetic modification that increases theexpression of a dicarboxylic acid transporter.
 12. The recombinant cellof claim 1 that, relative to a wild-type cell of identical cell type,produces a dicarboxylic acid not produced by the wild-type cell.
 13. Therecombinant cell of claim 1 that is a yeast cell.
 14. The recombinantcell of claim 13 selected from the group consisting of Bebaromyces,Candida, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia cells.15. The recombinant cell of claim 14 that is a Saccharomyces cell. 16.The recombinant cell of claim 15 that is S. cerevisiae.
 17. Therecombinant cell of claim 16 that contains a recombinant biotinbiosynthesis pathway and a recombinant BioI gene and produces pimelicacid.
 18. The recombinant cell of claim 16 that contains a recombinantType I fatty acid biosynthesis pathway and a recombinant fatty acidomega hydroxylase gene and produces adipic acid.
 19. The recombinantcell of claim 18 wherein the recombinant Type I fatty acid biosynthesispathway includes HexA and HexB genes.